TNF-alpha variants proteins for the treatment of TNF-alpha related disorders

ABSTRACT

The invention relates to novel proteins with TNF-alpha antagonist activity and nucleic acids encoding these proteins. The invention further relates to the use of the novel proteins in the treatment of TNF-alpha related disorders.

This application is a continuation-in-part of U.S. Ser. No. 09/981,289,filed Oct. 15, 2001 now U.S. Pat. No. 7,101,974; U.S. Ser. No.09/945,150, filed Aug. 31, 2001 now abandoned; and U.S. Ser. No.09/798,789, filed Mar. 2, 2001 now U.S. Pat. No. 7,056,695, which claimsthe benefit of the filing date of U.S. Ser. No. 60/186,427, filed Mar.2, 2000.

FIELD OF THE INVENTION

The invention relates to novel proteins with TNF-alpha antagonistactivity and nucleic acids encoding these proteins. The inventionfurther relates to the use of the novel proteins in the treatment ofTNF-alpha related disorders, such as autoimmune conditions, such asrheumatoid arthritis, sepsis and Crohn's disease, as well as peripheralnerve injury and demyelinating disorders.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNF-α or TNF-alpha) is a pleiotropic cytokinethat is primarily produced by activated macrophages and lymphocytes; butis also expressed in endothelial cells and other cell types. TNF-alphais a major mediator of inflammatory, immunological, andpathophysiological reactions. (Grell, M., et al., (1995) Cell,83:793-802). Two distinct forms of TNF exist, a 26 kDa membraneexpressed form and the soluble 17 kDa cytokine which is derived fromproteolytic cleavage of the 26 kDa form. The soluble TNF polypeptide is157 amino acids long and is the primary biologically active molecule.

TNF-alpha exerts its biological effects through interaction withhigh-affinity cell surface receptors. Two distinct membrane TNF-alphareceptors have been cloned and characterized. These are a 55 kDaspecies, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R(Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840). The twoTNF receptors exhibit 28% similarity at the amino acid level. This isconfined to the extracellular domain and consists of four repeatingcysteine-rich motifs, each of approximately 40 amino acids. Each motifcontains four to six cysteines in conserved positions. Dayhoff analysisshows the greatest intersubunit similarity among the first three repeatsin each receptor. This characteristic structure is shared with a numberof other receptors and cell surface molecules, which comprise theTNF-R/nerve growth factor receptor superfamily (Corcoran. A. E., et al.,(1994) Eur. J. Biochem., 223:831-840).

TNF signaling is initiated by receptor clustering, either by thetrivalent ligand TNF or by cross-linking monoclonal antibodies(Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627-1638).Crystallographic studies of TNF and the structurally related cytokine,lymphotoxin (LT) have shown that both cytokines exist as homotrimers,with subunits packed edge to edge in a threefold symmetry. Structurally,neither TNF or LT reflect the repeating pattern of the their receptors.Each monomer is cone shaped and contains two hydrophilic loops onopposite sides of the base of the cone. Recent crystal structuredetermination of a p55 soluble TNF-R/LT complex has confirmed thehypothesis that loops from adjacent monomers join together to form agroove between monomers and that TNF-R binds in these grooves (Corcoran.A. E., et al., (1994) Eur. J. Biochem., 223:831-840).

The key role played by TNF-alpha in inflammation, cellular immuneresponses and the pathology of many diseases has led to the search forantagonists of TNF-alpha. Soluble TNF receptors which interfere withTNF-alpha signaling have been isolated and are marketed by Immunex asEnbrel® for the treatment of rheumatoid arthritis. Random mutagenesishas been used to identify active sites in TNF-alpha responsible for theloss of cytotoxic activity (Van Ostade, X., et al., (1991) EMBO J.,10:827-836). Human TNF muteins having higher binding affinity for humanp75-TNF receptor than for human p55-TNF receptor have also beendisclosed (U.S. Pat. No. 5,597,899 and Loetscher et al., J. Biol. Chem.,268(35) pp263050-26357 (1993)). However, a need still exists to developmore potent TNF-alpha antagonists for use as therapeutic agents.

Accordingly, it is an object of the invention to provide proteins withTNF-alpha antagonist activity and nucleic acids encoding these proteinsfor the treatment of TNF-alpha related disorders.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides non-naturally occurring variant TNF-alpha proteins (e.g.proteins not found in nature) comprising amino acid sequences with atleast one amino acid change compared to the wild type TNF-alphaproteins.

Preferred embodiments utilize variant TNF-alpha proteins that interactwith the wild type TNF-alpha to form mixed trimers incapable ofactivating receptor signaling. Preferably, variant TNF-alpha proteinswith 1, 2, 3, 4, and 5 amino acid changes are used as compared to wildtype TNF-alpha protein. In a preferred embodiment, these changes areselected from positions 21, 23, 30, 31, 32, 33, 34, 35, 57, 65, 66, 67,75, 84, 86, 87, 91, 97, 111, 112, 115, 140, 143, 144, 145, 146 and 147.In an additional aspect, the non-naturally occurring variant TNF-alphaproteins have substitutions selected from the group of substitutionsconsisting of Q21C, Q21R, E23C, N34E, V91E, Q21R, N30D, R31C, R31I,R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C, L57F, L57W,L57Y, K65D, K65E, K65I, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K,G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, L75E, L75K, L75Q, A84V, S86Q,S86R, Y87H, Y87R, V91E, I97R, I97T, A111R, A111E, K112D, K112E, Y115D,Y115E, Y115F, Y115H, Y115I, Y115K, Y115L, Y115M, Y115N, Y115Q, Y115R,Y115S, Y115T, Y115W, D140K, D140R, D143E, D143K, D143L, D143R, D143N,D143Q, D143R, D143S, F144N, A145D, A145E, A145F, A145H, A145K, A145M,A145N, A145Q, A145R, A145S, A145T, A145Y, E146K, E146L, E146M, E146N,E146R, E146S and S147R.

In another preferred embodiment, substitutions may be made eitherindividually or in combination, with any combination being possible.Preferred embodiments utilize at least one, and preferably more,positions in each variant TNF-alpha protein. For example, substitutionsat positions 57, 75, 86, 87, 97, 115, 143, 145, and 146 may combined toform double variants. In addition triple point variants may begenerated.

In an alternative embodiment, non-naturally occurring TNF-alpha variantsin the form of monomers or dimers bind to the receptor interface todisrupt the binding of the wild-type TNF-alpha.

In a further embodiment, the TNF-alpha molecule may be chemicallymodified, for example by PEGylation or glycosylation.

In another aspect, portions of the N- or C-termini may deleted.

In a further embodiment, a TNF-alpha molecule may be circularlypermuted. In an additional aspect, the two or more receptor interactiondomains of the naturally occurring TNF-alpha or the TNF-alpha variantproteins are covalently linked by a linker peptide or by other means.

In a further aspect, the invention provides recombinant nucleic acidsencoding the non-naturally occurring variant TNF-alpha proteins,expression vectors, and host cells.

In an additional aspect, the invention provides methods of producing anon-naturally occurring variant TNF-alpha protein comprising culturingthe host cell of the invention under conditions suitable for expressionof the nucleic acid.

In a further aspect, the invention provides pharmaceutical compositionscomprising a variant TNF-alpha protein of the invention and apharmaceutical carrier.

In a further aspect, the invention provides methods for treating anTNF-alpha related disorder comprising administering a variant TNF-alphaprotein of the invention to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the design strategy for TNF-alpha mutants. FIG. 1Adepicts a complex of TNF receptor with wild type TNF-alpha. FIG. 1Bdepicts a mixed trimer of mutant TNF-alpha (TNF-X) and wild typeTNF-alpha. Dark circles are receptor molecules, light pentagons are wildtype TNF-alpha and the dark pentagon is a mutant TNF-alpha.

FIG. 2 depicts the structure of the wild type TNF-TNF-R trimer complex.

FIG. 3 depicts the structure of the p55 TNF-R extra-cellular domain. Thedarker appearing regions represent residues required for contact withTNF-alpha.

FIG. 4 depicts the binding sites on TNF-alpha that are involved inbinding the TNF-R.

FIG. 5 depicts the TNF-alpha trimer interface.

FIG. 6A depicts the nucleotide sequence (SEQ ID NO:1) of the histidinetagged wild type TNF-alpha molecule used as a template molecule fromwhich the mutants were generated. The additional 6 histidines, locatedbetween the start codon and the first amino acid are underlined.

FIG. 6B depicts the amino acid sequence (SEQ ID NO:2) of wild typeTNF-alpha with an additional 6 histidines (underlined) between the startcodon and the first amino acid. Amino acids changed in the TNF-alphamutants are shown in bold.

FIG. 7 depicts the position and the amino acid changes in the TNF-alphamutants (SEQ ID NOS:3-24).

FIG. 8 depicts the results from a TNF-alpha activity assay. Only one ofthe 11 TNF-alpha variants tested, E146K, was found to have agonisticactivity similar to wild-type TNF-alpha.

FIG. 9 depicts the antagonist activities of the TNF-alpha variants. Theresults shown are raw data that have not been normalized as a percent ofthe control. In this experiment, wild type TNF-alpha was used at 10ng/mL The concentration of the variant TNF-alpha proteins ranged from 1ng/mL to 50 μg/mL.

FIGS. 10A and 10B depicts the antagonist activities of the TNF-alphavariants normalized for percent apoptosis of the control.

FIG. 11 depicts another example of the mutation pattern of TNF-alphaprotein sequences. The probability table shows only the amino acidresidues of positions 21, 30, 31, 32, 33, 35, 65, 66, 67, 111, 112, 115,140, 143, 144, 145, 146 and 147. The occurrence of each amino acidresidue at a given position is indicated as a relative probability. Forexample, at position 21, the wild type amino acid is glutamine; in theTNF-alpha variants, arginine is the preferred amino acid at thisposition.

FIGS. 12A-F depicts trimerization domains from TRAF proteins (SEQ IDNO:25-30).

FIG. 13 depicts the synthesis of a full-length gene and all possiblemutations by PCR. Overlapping oligonucleotides corresponding to thefull-length gene (black bar, Step 1) and comprising one or more desiredmutations are synthesized, heated and annealed. Addition of DNApolymerase to the annealed oligonucleotides results in the 5′ to 3′synthesis of DNA (Step 2) to produce longer DNA fragments (Step 3).Repeated cycles of heating, annealing, and DNA synthesis (Step 4) resultin the production of longer DNA, including some full-length molecules.These can be selected by a second round of PCR using primers (indicatedby arrows) corresponding to the end of the full-length gene (Step 5).

FIG. 14 depicts a preferred method for synthesizing a library of thevariant TNF-alpha proteins of the invention using the wild-type gene.

FIG. 15 depict an overlapping extension method. At the top of FIG. 15Ais the template DNA showing the locations of the regions to be mutated(black boxes) and the binding sites of the relevant primers (arrows).The primers R1 and R2 represent a pool of primers, each containing adifferent mutation; as described herein, this may be done usingdifferent ratios of primers if desired. The variant position is flankedby regions of homology sufficient to get hybridization. In this example,three separate PCR reactions are done for step 1. The first reactioncontains the template plus oligos F1 and R1. The second reactioncontains template plus F2 and R2, and the third contains the templateand F3 and R3. The reaction products are shown. In Step 2, the productsfrom Step 1 tube 1 and Step 1 tube 2 are taken. After purification awayfrom the primers, these are added to a fresh PCR reaction together withF1 and R4. During the denaturation phase of the PCR, the overlappingregions anneal and the second strand is synthesized. T he product isthen amplified by the outside primers. In Step 3, the purified productfrom Step 2 is used in a third PCR reaction, together with the productof Step 1, tube 3 and the primers F1 and R3. The final productcorresponds to the full-length gene and contains the required mutations.

FIG. 16 depict a ligation of PCR reaction products to synthesize thelibraries of the invention. In this technique, the primers also containan endonuclease restriction site (RE), either blunt, 5′ overhanging or3′ overhanging. We set up three separate PCR reactions for Step 1. Thefirst reaction contains the template plus oligos F1 and R1. The secondreaction contains the template plus F2 and R2, and the third containsthe template and F3 and R3. The reaction products are shown. In Step 2,the products of step 1 are purified and then digested with theappropriate restriction endonuclease. The digestion products from Step2, tube 1 and Step 2, tube 2 and ligate them together with DNA ligase(step 3). The products are then amplified in Step 4 using primer F1 andR4. The whole process is then repeated by digesting the amplifiedproducts, ligating them to the digested products of Step 2, tube 3, andamplifying the final product by primers F1 and R3. It would also bepossible to ligate all three PCR products from Step 1 together in onereaction, providing the two restriction sites (RET and RE2) weredifferent.

FIG. 17 depicts blunt end ligation of PCR products. In this technique,the primers such as F1 and R1 do not overlap, but they abut. Again threeseparate PCR reactions are performed. The products from tube 1 and tube2 are ligated, and then amplified with outside primers F1 and R4. Thisproduct is then ligated with the product from Step 1, tube 3. The finalproducts are then amplified with primers F1 and R3.

FIG. 18 is a graphical illustration of the approach of identifyingchemical modification sites of the wild type TNF-alpha molecule.

FIGS. 19A-D depict the results of a TNFR1 binding assay of wild typeTNF-alpha and certain variants of the present invention.

FIG. 20A is a chart showing that the TNF-alpha variants of the presentinvention are pre-exchanged with wild type TNF-alpha to reduce TNF-alphainduced activation of NFkB in 293T cells. FIG. 20B are photographs ofthe immuno-localization of NFkB in HeLa cells showing that the exchangeof wild type TNF-alpha with the A145/Y87H TNF-alpha variant inhibitsTNF-alpha-induced nuclear translocation of NFkB in HeLa cells. FIG. 20Cdepicts the TNF-alpha variant A145R/Y87H reduces wild typeTNF-alpha-induced Activation of the NFkB-driven luciferase reporter.

FIG. 21 is a chart showing antagonist activity of TNF-alpha variants.

FIGS. 22A-C are dose response curves of caspase activation by variousTNF

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel proteins and nucleic acidspossessing TNF-alpha antagonist activity. The proteins are generatedusing a system previously described in WO98/47089 and U.S. Ser. Nos.09/058,459, 09/127,926, 60/104,612, 60/158,700, 09/419,351, 60/181,630,60/186,904, 09/419,351, 09/782,004 and 09/927,790, 60/347,772, and10/218,102, all of which are expressly incorporated by reference intheir entirety. In general, these applications describe a variety ofcomputational modeling systems that allow the generation of extremelystable proteins. In this way, variants of TNF proteins are generatedthat act as antagonists for wild type TNF-alpha. Variant TNF-proteinsmay be generated from wild type TNF-alpha, p55 TNF-R and p75 TNF-Rproteins, with preferred embodiments including variant TNF-alphaproteins.

Generally, there are a variety of computational methods that can be usedto generate a library of primary variant sequences. In a preferredembodiment, sequence-based methods are used. Alternatively,structure-based methods, such as the PDA™ technology, described indetail below, are used. Other models for assessing the relative energiesof sequences with high precision include Warshel, Computer Modeling ofChemical Reactions in Enzymes and Solutions, Wiley & Sons, New York,(1991), as well as the models identified in U.S. Ser. No. 10/218,102,filed Aug. 12, 2002, all hereby expressly incorporated by reference.

Similarly, molecular dynamics calculations can be used tocomputationally screen sequences by individually calculating mutantsequence scores and compiling a rank-ordered list.

In a preferred embodiment, residue pair potentials can be used to scoresequences (Miyazawa et al., Macromolecules 18(3):534-552 (1985),expressly incorporated by reference) during computational screening.

In a preferred embodiment, sequence profile scores (Bowie et al.,Science 253(5016):164-70 (1991), incorporated by reference) and/orpotentials of mean force (Hendlich et al., J. Mol. Biol. 216(1):167-180(1990), also incorporated by reference) may also be calculated to scoresequences. These methods assess the match between a sequence and a 3-Dprotein structure and hence can act to screen for fidelity to theprotein structure. By using different scoring functions to ranksequences, different regions of sequence space can be sampled in thecomputational screen.

Furthermore, scoring functions may be used to screen for sequences thatwould create metal or co-factor binding sites in the protein (Hellinga,Fold Des. 3(1): R1-8 (1998), hereby expressly incorporated byreference). Similarly, scoring functions may be used to screen forsequences that would create disulfide bonds in the protein. Thesepotentials attempt to specifically modify a protein structure tointroduce a new structural motif.

In a preferred embodiment, sequence and/or structural alignment programsmay be used to generate the variant TNF-alpha proteins of the invention.As is known in the art, there are a number of sequence-based alignmentprograms; including for example, Smith-Waterman searches,Needleman-Wunsch, Double Affine Smith-Waterman, frame search,Gribskov/GCG profile search, Gribskov/GCG profile scan, profile framesearch, Bucher generalized profiles, Hidden Markov models, Hframe,Double Frame, Blast, Psi-Blast, Clustal, and GeneWise.

The source of the sequences may vary widely, and include takingsequences from one or more of the known databases, including, but notlimited to, SCOP (Hubbard, et al., Nucleic Acids Res 27(1):254-256.(1999)); PFAM (Bateman, et al., Nucleic Acids Res 27(1):260-262.(1999)); VAST (Gibrat, et al., Curr Opin Struct Biol 6(3):377-385.(1996)); CATH (Orengo, et al., Structure 5(8):1093-1108. (1997)); PhDPredictor; Prosite (Hofmann, et al., Nucleic Acids Res 27(1):215-219.(1999)); PIR; GenBank; PDB and BIND (Bader, et al., Nucleic Acids Res29(1):242-245. (2001)).

In addition, sequences from these databases may be subjected tocontiguous analysis or gene prediction; see Wheeler, et al., NucleicAcids Res 28(1):10-14. (2000) and Burge and Karlin, J Mol Biol268(1):78-94. (1997).

As is known in the art, there are a number of sequence alignmentmethodologies that may be used. For example, sequence homology basedalignment methods may be used to create sequence alignments of proteinsrelated to the target structure (Altschul et al., J. Mol. Biol.215(3):403-410 (1990), Altschul et al., Nucleic Acids Res. 25:3389-3402(1997), both incorporated by reference). These sequence alignments arethen examined to determine the observed sequence variations. Thesesequence variations are tabulated to define a set of variant TNF-alphaproteins.

Sequence based alignments may be used in a variety of ways. For example,a number of related proteins may be aligned, as is known in the art, andthe “variable” and “conserved” residues defined; that is, the residuesthat vary or remain identical between the family members can be defined.These results may be used to generate a probability table, as outlinedbelow. Similarly, these sequence variations may be tabulated and asecondary library defined from them as defined below. Alternatively, theallowed sequence variations may be used to define the amino acidsconsidered at each position during the computational screening. Anothervariation, is to bias the score for amino acids that occur in thesequence alignment, thereby increasing the likelihood that they arefound during computational screening but still allowing consideration ofother amino acids. This bias would result in a focused library ofvariant TNF-alpha proteins but would not eliminate from considerationamino acids not found in the alignment. In addition, a number of othertypes of bias may be introduced. For example, diversity may be forced;that is, a “conserved” residue is chosen and altered to force diversityon the protein and thus sample a greater portion of the sequence space.Alternatively, the positions of high variability between family members(i.e. low conservation) may be randomized, either using all or a subsetof amino acids. Similarly, outlier residues, either positional outliersor side chain outliers, may be eliminated.

Similarly, structural alignment of structurally related proteins may bedone to generate sequence alignments. There are a wide variety of suchstructural alignment programs known. See for example VAST from the NCBIwebsite; SSAP (Orengo and Taylor, Methods Enzymol 266(617-635 (1996))SARF2 (Alexandrov, Protein Eng 9(9):727-732. (1996)) CE (Shindyalov andBourne, Protein Eng 11(9):739-747. (1998)); (Orengo et al., Structure5(8):1093-108 (1997); Dali (Holm et al., Nucleic Acid Res. 26(1):316-9(1998), all of which are incorporated by reference). These sequencealignments may then be examined to determine the observed sequencevariations. Libraries may be generated by predicting secondary structurefrom sequence, and then selecting sequences that are compatible with thepredicted secondary structure. There are a number of secondary structureprediction methods such as helix-coil transition theory (Munoz andSerrano, Biopolymers 41:495, 1997), neural networks, local structurealignment and others (e.g., see in Selbig et al., Bioinformatics15:1039-46, 1999).

Similarly, as outlined above, other computational methods are known,including, but not limited to, sequence profiling [Bowie and Eisenberg,Science 253(5016):164-70, (1991)], rotamer library selections [Dahiyatand Mayo, Protein Sci. 5(5):895-903 (1996); Dahiyat and Mayo, Science278(5335):82-7 (1997); Desjarlais and Handel, Protein Science4:2006-2018 (1995); Harbury et al, Proc. Natl. Acad. Sci. U.S.A.92(18):8408-8412 (1995); Kono et al., Proteins: Structure, Function andGenetics 19:244-255 (1994); Hellinga and Richards, Proc. Natl. Acad.Sci. U.S.A. 91:5803-5807 (1994)]; and residue pair potentials [Jones,Protein Science 3: 567-574, (1994)]; PROSA [Heindlich et al., J. Mol.Biol. 216:167-180 (1990; THREADER [Jones et al., Nature 358:86-89(1992)], and other inverse folding methods such as those described bySimons et al. [Proteins, 34:535-543, (1999)], Levitt and Gerstein [Proc.Natl. Acad. Sci. U.S.A., 95:5913-5920, (1998)], Godzik and Skolnick[Proc. Natl. Acad. Sci. U.S.A., 89:12098-102, (1992)], Godzik et al. [J.Mol. Biol. 227:227-38, (1992)] and two profile methods [Gribskov et al.Proc. Natl. Acad. Sci. U.S.A. 84:4355-4358 (1987) and Fischer andEisenberg, Protein Sci. 5:947-955 (1996), Rice and Eisenberg J. Mol.Biol. 267:1026-1038(1997)], all of which are expressly incorporated byreference.

In addition, other computational methods such as those described byKoehl and Levitt (J. Mol. Biol. 293:1161-1181 (1999); J. Mol. Biol.293:1183-1193 (1999); expressly incorporated by reference) may be usedto create a variant TNF-alpha library which may optionally then be usedto generate a smaller secondary library for use in experimentalscreening for improved properties and function. In addition, there arecomputational methods based on force field calculations such as SCMF,see Delarue et al. Pac. Symp. Biocomput. 109-21 (1997); Koehl et al., J.Mol. Biol. 239:249-75 (1994); Koehl et al., Nat. Struct. Biol. 2:163-70(1995); Koehl et al., Curr. Opin. Struct. Biol. 6:222-6 (1996); Koehl etal., J. Mol. Biol. 293:1183-93 (1999); Koehl et al., J. Mol. Biol.293:1161-81 (1999); Lee J., Mol. Biol. 236:918-39 (1994); and VasquezBiopolymers 36:53-70 (1995); all of which are expressly incorporated byreference. Other force field calculations that can be used to optimizethe conformation of a sequence within a computational method, or togenerate de novo optimized sequences as outlined herein include, but arenot limited to, OPLS-AA [Jorgensen et al., J. Am. Chem. Soc.118:11225-11236 (1996); Jorgensen, W. L.; BOSS, Version 4.1; YaleUniversity: New Haven, Conn. (1999)]; OPLS [Jorgensen et al., J. Am.Chem. Soc. 110:1657ff (1988); Jorgensen et al., J. Am. Chem. Soc.112:4768ff (1990)]; UNRES (United Residue Forcefield; Liwo et al.,Protein Science 2:1697-1714 (1993); Liwo et al., Protein Science2:1715-1731 (1993); Liwo et al., J. Comp. Chem. 18:849-873 (1997); Liwoet al., J. Comp. Chem. 18:874-884 (1997); Liwo et al., J. Comp. Chem.19:259-276 (1998); Forcefield for Protein Structure Prediction (Liwo etal., Proc. Natl. Acad. Sci. U.S.A. 96:5482-5485 (1999)]; ECEPP/3 [Liwoet al., J Protein Chem. 13(4):375-80 (1994)]; AMBER 1.1 force field(Weiner et al., J. Am. Chem. Soc. 106:765-784); AMBER 3.0 force field[U. C. Singh et al., Proc. Natl. Acad. Sci. U.S.A. 82:755-759 (1985)];CHARMM and CHARMM22 (Brooks et al., J. Comp. Chem. 4:187-217); cvff3.0[Dauber-Osguthorpe et al., Proteins: Structure, Function and Genetics,4:31-47 (1988)]; cff91 (Maple et al., J. Comp. Chem. 15:162-182); also,the DISCOVER (cvff and cff91) and AMBER forcefields are used in theINSIGHT molecular modeling package (Biosym/MSI, San Diego Calif.) andHARMM is used in the QUANTA molecular modeling package (Biosym/MSI, SanDiego Calif.), all of which are expressly incorporated by reference. Infact, as is outlined below, these force field methods may be used togenerate the variant TNF-alpha library directly; these methods may beused to generate a probability table from which an additional library isdirectly generated.

In a preferred embodiment, the computational method used to generate theset or library of variant TNF-alpha proteins is Protein DesignAutomation™ (PDA™) technology, as is described in U.S. Ser. Nos.60/061,097, 60/043,464, 60/054,678, 09/127,926, 60/104,612, 60/158,700,09/419,351, 60/181,630, 60/186,904, 09/419,351, 09/782,004, 09/927,790,and PCT US98/07254, and Ser. No. 10/218,102, all of which are expresslyincorporated herein by reference.

PDA™ technology uses a known protein structure as a starting point. Theresidues to be optimized are then identified, which may be the entiresequence or subset(s) thereof. The side chains of any positions to bevaried are then removed. The resulting structure consisting of theprotein backbone and the remaining side chains is called the template.Each variable residue position may optionally be classified as a coreresidue, a surface residue, or a boundary residue; each classificationdefines a subset of possible amino acid residues for the position (forexample, core residues generally will be selected from the set ofhydrophobic residues, surface residues generally will be selected fromthe hydrophilic residues, and boundary residues may be either). Eachamino acid can be represented by a discrete set of all allowedconformers of each side chain, called rotamers. Thus, to arrive at anoptimal sequence for a backbone, all possible sequences of rotamers mustbe screened, where each backbone position may be occupied either by eachamino acid in all its possible rotameric states, or a subset of aminoacids, and thus a subset of rotamers.

Two sets of interactions are then calculated for each rotamer at everyposition: the interaction of the rotamer side chain with all or part ofthe backbone (the “singles” energy, also called the rotamer/template orrotamer/backbone energy), and the interaction of the rotamer side chainwith all other possible rotamers at every other position or a subset ofthe other positions (the “doubles” energy, also called therotamer/rotamer energy). The energy of each of these interactions iscalculated through the use of a variety of scoring functions, whichinclude the energy of van der Waal's forces, the energy of hydrogenbonding, the energy of secondary structure propensity, the energy ofsurface area solvation and the electrostatics. Thus, the total energy ofeach rotamer interaction, both with the backbone and other rotamers, iscalculated, and stored in a matrix form.

The discrete nature of rotamer sets allows a simple calculation of thenumber of rotamer sequences to be tested. A backbone of length n with mpossible rotamers per position will have mn possible rotamer sequences,a number which grows exponentially with sequence length and renders thecalculations either unwieldy or impossible in real time. Accordingly, tosolve this combinatorial search problem, various algorithms may beemployed. For example, a “Dead End Elimination” (DEE) calculation may beperformed. The DEE calculation is based on the fact that if the worsttotal interaction of a first rotamer is still better than the best totalinteraction of a second rotamer, then the second rotamer cannot be partof the global optimum solution. Since the energies of all rotamers havealready been calculated, the DEE approach only requires sums over thesequence length to test and eliminate rotamers, which speeds up thecalculations considerably. DEE can be rerun comparing pairs of rotamers,or combinations of rotamers, which will eventually result in thedetermination of a single sequence which represents the global optimumenergy.

Once the global solution has been found, a Monte Carlo search may bedone to generate a rank-ordered list or filtered set of sequences in theneighborhood of the DEE solution. Starting at the DEE solution, randompositions are changed to other rotamers, and the new sequence energy iscalculated. If the new sequence meets the criteria for acceptance, it isused as a starting point for another jump. After a predetermined numberof jumps, a rank-ordered list of sequences is generated. Monte Carlosearching is a sampling technique to explore sequence space around theglobal minimum or to find new local minima distant in sequence space. Asis more additionally outlined below, there are other sampling techniquesthat may be used, including Boltzmann sampling, genetic algorithmtechniques and simulated annealing. In addition, for all the samplingtechniques, the kinds of jumps allowed may be altered (e.g. random jumpsto random residues, biased jumps (to or away from wild type, forexample), jumps to biased residues (to or away from similar residues,for example), etc.). Similarly, for all the sampling techniques, theacceptance criteria of whether a sampling jump is accepted may bealtered.

As outlined in U.S. Ser. No. 09/127,926, now U.S. Pat. No. 6,269,312,and Ser. No. 10/218,102, the protein backbone comprising (for anaturally occurring protein) the nitrogen, the carbonyl carbon, theα-carbon, and the carbonyl oxygen, along with the direction of thevector from the α-carbon to the, β-carbon may be altered prior to thecomputational analysis, by varying a set of parameters calledsupersecondary structure parameters.

Once a protein structure backbone is generated (with alterations, asoutlined above) and input into the computer, explicit hydrogens areadded if not included within the structure (for example, if thestructure was generated by X-ray crystallography, hydrogens must beadded). After hydrogen addition, energy minimization of the structure isrun, to relax the hydrogens as well as the other atoms, bond angles andbond lengths. In a preferred embodiment, this is done by doing a numberof steps of conjugate gradient minimization [Mayo et al., J. Phys. Chem.94:8897 (1990)] of atomic coordinate positions to minimize the Dreidingforce field with no electrostatics. Generally, from about 10 to about250 steps is preferred, with about 50 being most preferred.

The protein backbone structure contains at least one variable residueposition. As is known in the art, the residues, or amino acids, ofproteins are generally sequentially numbered starting with theN-terminus of the protein. Thus a protein having a methionine at it'sN-terminus is said to have a methionine at residue or amino acidposition 1, with the next residues as 2, 3, 4, etc. At each position,the wild-type (i.e. naturally occurring) protein may have one of atleast 20 amino acids, in any number of rotamers. By “variable residueposition” herein is meant an amino acid position of the protein to bedesigned that is not fixed in the design method as a specific residue orrotamer, generally the wild type residue or rotamer.

In a preferred embodiment, all of the residue positions of the proteinare variable. That is, every amino acid side chain may be altered in themethods of the present invention. This is particularly desirable forsmaller proteins, although the present methods allow the design oflarger proteins as well. While there is no theoretical limit to thelength of the protein which may be designed this way, there is apractical computational limit.

In an alternate preferred embodiment, only some of the residue positionsof the protein are variable, and the remainder are “fixed”, that is,they are identified in the three dimensional structure as being in a setconformation. In some embodiments, a fixed position is left in itsoriginal conformation (which may or may not correlate to a specificrotamer of the rotamer library being used). Alternatively, residues maybe fixed as a non-wild type residue; for example, when knownsite-directed mutagenesis techniques have shown that a particularresidue is desirable (for example, to eliminate a proteolytic site oralter the substrate specificity of an enzyme), the residue may be fixedas a particular amino acid. Alternatively, the methods of the presentinvention may be used to evaluate mutations de novo, as is discussedbelow. In an alternate preferred embodiment, a fixed position may be“floated”; the amino acid at that position is fixed, but differentrotamers of that amino acid are tested. In this embodiment, the variableresidues may be at least one, or anywhere from 0.1% to 99.9% of thetotal number of residues. Thus, for example, it may be possible tochange only a few (or one) residues, or most of the residues, with allpossibilities in between.

In a preferred embodiment, residues which can be fixed include, but arenot limited to, structurally or biologically functional residues;alternatively, biologically functional residues may specifically not befixed. For example, residues which are known to be important forbiological activity, such as the residues which the binding site for abinding partner (ligand/receptor, antigen/antibody, etc.),phosphorylation or glycosylation sites which are crucial to biologicalfunction, or structurally important residues, such as disulfide bridges,metal binding sites, critical hydrogen bonding residues, residuescritical for backbone conformation such as proline or glycine, residuescritical for packing interactions, etc. may all be fixed in aconformation or as a single rotamer, or “floated”.

Similarly, residues which may be chosen as variable residues may bethose that confer undesirable biological attributes, such assusceptibility to proteolytic degradation, dimerization or aggregationsites, glycosylation sites which may lead to immune responses, unwantedbinding activity, unwanted allostery, undesirable enzyme activity butwith a preservation of binding, etc. In the present invention, it is thetetramerization domain residues which are varied, as outlined below.

In an alternative embodiment, each variable position is classified as acore, surface or boundary residue position, although in some cases, asexplained below, the variable position may be set to glycine to minimizebackbone strain. In addition, as outlined herein, residues need not beclassified, they can be chosen as variable and any set of amino acidsmay be used. Any combination of core, surface and boundary positions canbe utilized: core, surface and boundary residues; core and surfaceresidues; core and boundary residues, and surface and boundary residues,as well as core residues alone, surface residues alone, or boundaryresidues alone.

The classification of residue positions as core, surface or boundary maybe done in several ways, as will be appreciated by those in the art. Ina preferred embodiment, the classification is done via a visual scan ofthe original protein backbone structure, including the side chains, andassigning a classification based on a subjective evaluation of oneskilled in the art of protein modeling. Alternatively, a preferredembodiment utilizes an assessment of the orientation of the Cα-Cβvectors relative to a solvent accessible surface computed using only thetemplate Cα atoms, as outlined in U.S. Ser. Nos. 60/061,097, 60/043,464,60/054,678, 09/127,926, now U.S. Pat. No. 6,269,312, 60/104,612,60/158,700, Ser. Nos. 09/419,351, 60/181,630, 60/186,904, 09/419,351,now U.S. Pat. No. 6,403,312, Ser. Nos. 09/782,004 and 09/927,790, filedAug. 10, 2001, PCT US98/07254 and Ser. No. 10/218,102. Alternatively, asurface area calculation can be done.

Once each variable position is classified as core, surface or boundary,a set of amino acid side chains, and thus a set of rotamers, is assignedto each position. That is, the set of possible amino acid side chainsthat the program will allow to be considered at any particular positionis chosen. Subsequently, once the possible amino acid side chains arechosen, the set of rotamers that will be evaluated at a particularposition can be determined. Thus, a core residue will generally beselected from the group of hydrophobic residues consisting of alanine,valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, andmethionine (in some embodiments, when the α scaling factor of the vander Waals scoring function, described below, is low, methionine isremoved from the set), and the rotamer set for each core positionpotentially includes rotamers for these eight amino acid side chains(all the rotamers if a backbone independent library is used, and subsetsif a rotamer dependent backbone is used).

Similarly, surface positions are generally selected from the group ofhydrophilic residues consisting of alanine, serine, threonine, asparticacid, asparagine, glutamine, glutamic acid, arginine, lysine andhistidine. The rotamer set for each surface position thus includesrotamers for these ten residues. Finally, boundary positions aregenerally chosen from alanine, serine, threonine, aspartic acid,asparagine, glutamine, glutamic acid, arginine, lysine histidine,valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, andmethionine. The rotamer set for each boundary position thus potentiallyincludes every rotamer for these seventeen residues (assuming cysteine,glycine and proline are not used, although they can be). Additionally,in some preferred embodiments, a set of 18 naturally occurring aminoacids (all except cysteine and proline, which are known to beparticularly disruptive) are used.

Thus, as will be appreciated by those in the art, there is acomputational benefit to classifying the residue positions, as itdecreases the number of calculations. It should also be noted that theremay be situations where the sets of core, boundary and surface residuesare altered from those described above; for example, under somecircumstances, one or more amino acids is either added or subtractedfrom the set of allowed amino acids. For example, some proteins whichdimerize, trimerize or multimerize, or have ligand binding sites, maycontain hydrophobic surface residues, etc. In addition, residues that donot allow helix “capping” or the favorable interaction with an α-helixdipole may be subtracted from a set of allowed residues. Thismodification of amino acid groups is done on a residue by residue basis.

In a preferred embodiment, proline, cysteine and glycine are notincluded in the list of possible amino acid side chains, and thus therotamers for these side chains are not used. However, in a preferredembodiment, when the variable residue position has a φ angle that is,the dihedral angle defined by 1) the carbonyl carbon of the precedingamino acid; 2) the nitrogen atom of the current residue; 3) the α-carbonof the current residue; and 4) the carbonyl carbon of the currentresidue greater than 0 degrees, the position is set to glycine tominimize backbone strain.

Once the group of potential rotamers is assigned for each variableresidue position, processing proceeds as outlined in U.S. Ser. No.09/127,926, now U.S. Pat. No. 6,269,312, and PCT US98/07254 and Ser. No.10/218,102. This processing step entails analyzing interactions of therotamers with each other and with the protein backbone to generateoptimized protein sequences. Simplistically, the processing initiallycomprises the use of a number of scoring functions to calculate energiesof interactions of the rotamers, either to the backbone itself or otherrotamers. Preferred PDA scoring functions include, but are not limitedto, a Van der Waals potential scoring function, a hydrogen bondpotential scoring function, an atomic solvation scoring function, asecondary structure propensity scoring function and an electrostaticscoring function. As is further described below, at least one scoringfunction is used to score each position, although the scoring functionsmay differ depending on the position classification or otherconsiderations, like favorable interaction with an α-helix dipole. Asoutlined below, the total energy which is used in the calculations isthe sum of the energy of each scoring function used at a particularposition, as is generally shown in Equation 1:E _(total) =nE _(vdw) +nE _(as) +nE _(h-bonding) +nE _(ss) +nE_(elec)  Equation 1

In Equation 1, the total energy is the sum of the energy of the Van derWaals potential (E_(vdw)), the energy of atomic salvation (E_(as)), theenergy of hydrogen bonding (E_(h-bonding)), the energy of secondarystructure (E_(ss)) and the energy of electrostatic interaction(E_(elec)). The term n is either 0 or 1, depending on whether the termis to be considered for the particular residue position.

As outlined in U.S. Ser. Nos. 60/061,097, 60/043,464, 60/054,678,09/127,926, now U.S. Pat. No. 6,269,312, 60/104,612, 60/158,700,60/181,630, 60/186,904, 60/192,851, 09/418,719, 09/419,351, now U.S.Pat. No. 6,403,312, Ser. Nos. 09/782,004, 09/927,790, PCT US98/07254,Ser. Nos. 09/877,695, 10/071,859, 10/101,499 and 10/218,102, anycombination of these scoring functions, either alone or in combination,may be used.

Once the scoring functions to be used are identified for each variableposition, the preferred first step in the computational analysiscomprises the determination of the interaction of each possible rotameror amino acid with all or part of the remainder of the protein. That is,the energy of interaction, as measured by one or more of the scoringfunctions, of each possible rotamer or amino acid at each variableresidue position with either the backbone or other rotamers or aminoacids, is calculated. In a preferred embodiment, the interaction of eachrotamer or amino acid with the entire remainder of the protein, i.e.both the entire template and all other rotamers or amino acids, is done.However, as outlined above, it is possible to only model a portion of aprotein, for example a domain of a larger protein, and thus in somecases, not all of the protein need be considered. The term “portion”, orsimilar grammatical equivalents thereof, as used herein, with regard toa protein refers to a fragment of that protein. This fragment may rangein size from 6-10 amino acid residues to the entire amino acid sequenceminus one amino acid. Accordingly, the term “portion”, as used herein,with regard to a nucleic refers to a fragment of that nucleic acid. Thisfragment may range in size from 10 nucleotides to the entire nucleicacid sequence minus one nucleotide.

In a preferred embodiment, the first step of the computationalprocessing is done by calculating two sets of interactions for eachrotamer or amino acid at every position: the interaction of the rotamerside chain or amino acid with the template or backbone (the “singles”energy), and the interaction of the rotamer side chain with all otherpossible rotamers or amino acids at every other position (the “doubles”energy), whether that position is varied or floated. It should beunderstood that the backbone in this case includes both the atoms of theprotein structure backbone, as well as the atoms of any fixed residues,wherein the fixed residues are defined as a particular conformation ofan amino acid.

Thus, “singles” (rotamer/template) energies are calculated for theinteraction of every possible rotamer or amino acid at every variableresidue position with the backbone, using some or all of the scoringfunctions. Thus, for the hydrogen bonding scoring function, everyhydrogen bonding atom of the rotamer or amino acid and every hydrogenbonding atom of the backbone is evaluated, and the E_(HB) is calculatedfor each possible rotamer or amino acid at every variable position.Similarly, for the Van der Waals scoring function, every atom of therotamer or amino acid is compared to every atom of the template(generally excluding the backbone atoms of its own residue), and theE_(vdw) is calculated for each possible rotamer or amino acid at everyvariable residue position. In addition, generally no Van der Waalsenergy is calculated if the atoms are connected by three bonds or less.For the atomic solvation scoring function, the surface of the rotamer oramino acid is measured against the surface of the template, and theE_(as) for each possible rotamer or amino acid at every variable residueposition is calculated. The secondary structure propensity scoringfunction is also considered as a singles energy, and thus the totalsingles energy may contain an E_(ss) term. As will be appreciated bythose in the art, many of these energy terms will be close to zero,depending on the physical distance between the rotamer or amino acid andthe template position; that is, the farther apart the two moieties, thelower the energy.

For the calculation of “doubles” energy (e.g., rotamer/rotamer), theinteraction energy of each possible rotamer or amino acid is comparedwith every possible rotamer or amino acid at all other variable residuepositions. Thus, “doubles” energies are calculated for the interactionof every possible rotamer or amino acid at every variable residueposition with every possible rotamer or amino acid at every othervariable residue position, using some or all of the scoring functions.Thus, for the hydrogen bonding scoring function, every hydrogen bondingatom of the first rotamer or amino acid and every hydrogen bonding atomof every possible second rotamer or amino acid is evaluated, and theE_(HB) is calculated for each possible rotamer or amino acid pair forany two variable positions. Similarly, for the Van der Waals scoringfunction, every atom of the first rotamer or amino acid is compared toevery atom of every possible second rotamer or amino acid, and theE_(vdw) is calculated for each possible rotamer or amino acid pair atevery two variable residue positions. For the atomic solvation scoringfunction, the surface of the first rotamer or amino acid is measuredagainst the surface of every possible second rotamer or amino acid, andthe E_(as) for each possible rotamer or amino acid pair at every twovariable residue positions is calculated. The secondary structurepropensity scoring function need not be run as a “doubles” energy, as itis considered as a component of the “singles” energy. As will beappreciated by those in the art, many of these double energy terms willbe close to zero, depending on the physical distance between the firstrotamer and the second rotamer; that is, the farther apart the twomoieties, the lower the energy.

In addition, as will be appreciated by those in the art, a variety offorce fields that can be used in the PDA™ technology calculations may beused, including, but not limited to, Dreiding I and Dreiding II [Mayo etal, J. Phys. Chem. 94:8897 (1990)], AMBER [Weiner et al., J. Amer. Chem.Soc. 106:765 (1984) and Weiner et al., J. Comp. Chem. 106:230 (1986)],MM2 [Allinger, J. Chem. Soc. 99:8127 (1977), Liljefors et al., J. Com.Chem. 8:1051 (1987)]; MMP2 [Sprague et al., J. Comp. Chem. 8:581(1987)]; CHARMM [Brooks et al., J. Comp. Chem. 106:187 (1983)]; GROMOS;and MM3 [Allinger et al., J. Amer. Chem. Soc. 111:8551 (1989)], OPLS-AA[Jorgensen et al., J. Am. Chem. Soc. 118:11225-11236 (1996); Jorgensen,W. L.; BOSS, Version 4.1; Yale University: New Haven, Conn. (1999)];OPLS [Jorgensen et al., J. Am. Chem. Soc. 110:1657ff (1988); Jorgensenet al., J. Am. Chem. Soc. 112:4768ff (1990)]; UNRES (United ResidueForcefield; Liwo et al., Protein Science 2:1697-1714 (1993); Liwo etal., Protein Science 2:1715-1731 (1993); Liwo et al., J. Comp. Chem.18:849-873 (1997); Liwo et al., J. Comp. Chem. 18:874-884 (1997); Liwoet al., J. Comp. Chem. 19:259-276 (1998); Forcefield for ProteinStructure Prediction (Liwo et al., Proc. Natl. Acad. Sci. U.S.A96:5482-5485 (1999)]; ECEPP/3 [Liwo et al., J Protein Chem. 13(4):375-80(1994)]; AMBER 1.1 force field (Weiner, et al., J. Am. Chem. Soc.106:765-784); AMBER 3.0 force field (U. C. Singh et al., Proc. Natl.Acad. Sci. U.S.A. 82:755-759); CHARMM and CHARMM22 (Brooks et al., J.Comp. Chem. 4:187-217); cvff3.0 [Dauber-Osguthorpe, et al., Proteins:Structure, Function and Genetics, 4:31-47 (1988)]; cff91 (Maple, et al.,J. Comp. Chem. 15:162-182); also, the DISCOVER (cvff and cff91) andAMBER forcefields are used in the INSIGHT molecular modeling package(Biosym/MSI, San Diego Calif.) and HARMM is used in the QUANTA molecularmodeling package (Biosym/MSI, San Diego Calif.), all of which areexpressly incorporated by reference.

Once the singles and doubles energies are calculated and stored, thenext step of the computational processing may occur. As outlined in U.S.Ser. No. 09/127,926, now U.S. Pat. No. 6,269,312, PCT US98/07254 andU.S. Ser. No. 10/218,102, preferred embodiments may utilize a Dead EndElimination (DEE) step, and a Monte Carlo step.

The PDA™ technology, viewed broadly, has three components that may bevaried to alter the output (e.g. the primary library): the scoringfunctions used in the process; the filtering technique, and the samplingtechnique.

In a preferred embodiment, the scoring functions may be altered. In apreferred embodiment, the scoring functions outlined above may be biasedor weighted in a variety of ways. For example, a bias towards or awayfrom a reference sequence or family of sequences can be done; forexample, a bias towards wild type or homologue residues may be used.Similarly, the entire protein or a fragment of it may be biased; forexample, the active site may be biased towards wild type residues, ordomain residues towards a particular desired physical property can bedone. Furthermore, a bias towards or against increased energy can begenerated. Additional scoring function biases include, but are notlimited to applying electrostatic potential gradients or hydrophobicitygradients, adding a substrate or binding partner to the calculation, orbiasing towards a desired charge or hydrophobicity.

In addition, in an alternative embodiment, there are a variety ofadditional scoring functions that may be used. Additional scoringfunctions include, but are not limited to torsional potentials, orresidue pair potentials, or residue entropy potentials. Such additionalscoring functions can be used alone, or as functions for processing thelibrary after it is scored initially. For example, a variety offunctions derived from data on binding of peptides to MHC (MajorHistocompatibility Complex) may be used to rescore a library in order toeliminate proteins containing sequences, which can potentially bind toMHC, i.e. potentially immunogenic sequences. See, for example, U.S. Ser.No. 60/217,661; 09/903,378; 10/039,170; 60/360,843; 60/384,197; PCT01/21,823; and PCT 02/00165.

In a preferred embodiment, a variety of filtering techniques may bedone, including, but not limited to, DEE and its related counterparts.Additional filtering techniques include, but are not limited tobranch-and-bound techniques for finding optimal sequences (Gordon andMayo, Structure Fold. Des. 7:1089-98, 1999), and exhaustive enumerationof sequences. It should be noted however, that some techniques may alsobe done without any filtering techniques; for example, samplingtechniques can be used to find good sequences, in the absence offiltering.

As will be appreciated by those in the art, once an optimized sequenceor set of sequences is generated, a variety of sequence space samplingmethods can be done, either in addition to the preferred Monte Carlomethods, or instead of a Monte Carlo search. That is, once a sequence orset of sequences is generated, preferred methods utilize samplingtechniques to allow the generation of additional, related sequences fortesting.

These sampling methods can include the use of amino acid substitutions,insertions or deletions, or recombinations of one or more sequences. Asoutlined herein, a preferred embodiment utilizes a Monte Carlo search,which is a series of biased, systematic, or random jumps. However, thereare other sampling techniques that may be used, including Boltzmannsampling, genetic algorithm techniques and simulated annealing. Inaddition, for all the sampling techniques, the kinds of jumps allowedmay be altered (e.g. random jumps to random residues, biased jumps (toor away from wild type, for example), jumps to biased residues (to oraway from similar residues, for example, etc.). Jumps where multipleresidue positions are coupled (two residues always change together, ornever change together), jumps where whole sets of residues change toother sequences (e.g., recombination). Similarly, for all the samplingtechniques, the acceptance criteria of whether a sampling jump isaccepted may be altered, to allow broad searches at high temperature andnarrow searches close to local optima at low temperatures. SeeMetropolis et al., J. Chem Phys v21, pp 1087, 1953, hereby expresslyincorporated by reference.

In addition, it should be noted that the preferred methods of theinvention result in a rank-ordered or a filtered list of sequences; thatis, the sequences are ranked on the basis of some objective criteria.However, as outlined herein, it is possible to create a set ofnon-ordered sequences, for example by generating a probability tabledirectly (for example using SCMF analysis or sequence alignmenttechniques) that lists sequences without ranking them. The samplingtechniques outlined herein can be used in either situation.

In a preferred embodiment, Boltzmann sampling is done. As will beappreciated by those in the art, the temperature criteria for Boltzmannsampling can be altered to allow broad searches at high temperature andnarrow searches close to local optima at low temperatures (see e.g.,Metropolis et al., J. Chem. Phys. 21:1087, 1953).

In a preferred embodiment, the sampling technique utilizes geneticalgorithms, e.g., such as those described by Holland (Adaptation inNatural and Artificial Systems, 1975, Ann Arbor, U. Michigan Press).

Genetic algorithm analysis generally takes generated sequences andrecombines them computationally, similar to a nucleic acid recombinationevent, in a manner similar to “gene shuffling”. Thus the “jumps” ofgenetic algorithm analysis generally are multiple position jumps. Inaddition, as outlined below, correlated multiple jumps may also be done.Such jumps may occur with different crossover positions and more thanone recombination at a time, and may involve recombination of two ormore sequences.

Furthermore, deletions or insertions (random or biased) can be done. Inaddition, as outlined below, genetic algorithm analysis may also be usedafter the secondary library has been generated.

In a preferred embodiment, the sampling technique utilizes simulatedannealing, e.g., such as described by Kirkpatrick et al. [Science,220:671-680 (1983)]. Simulated annealing alters the cutoff for acceptinggood or bad jumps by altering the temperature. That is, the stringencyof the cutoff is altered by altering the temperature. This allows broadsearches at high temperature to new areas of sequence space, alteringwith narrow searches at low temperature to explore regions in detail.In addition, as outlined below, these sampling methods may be used tofurther process a first set to generate additional sets of variantTNF-alpha proteins.

As used herein variant TNF-alpha or TNF-alpha proteins include TNF-alpha(or TNF-α) monomers or dimers.

The computational processing results in a set of optimized variant TNFprotein sequences. Optimized variant TNF-alpha protein sequences aregenerally different from the wild type TNF-alpha sequence in structuralregions critical for receptor affinity, e.g. p55, p75 (see FIGS. 2-4).Preferably, each optimized variant TNF-alpha protein sequence comprisesat least about 1 variant amino acid from the starting or wild-typesequence, with 3-5 being preferred.

Thus, in the broadest sense, the present invention is directed tovariant TNF-alpha proteins that are antagonists of wild type TNF-alpha.By “variant TNF-alpha or TNF-α proteins” herein is meant TNF-alpha orTNF-α proteins, which have been designed using the computational methodsoutlined herein to differ from the corresponding wild type protein by atleast 1 amino acid.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures, i.e.,“analogs” such as peptoids [see Simon et al., Proc. Natl. Acd. Sci.U.S.A. 89(20:9367-71 (1992)], generally depending on the method ofsynthesis. Thus “amino acid”, or “peptide residue”, as used herein meansboth naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline, and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. In addition, anyamino acid representing a component of the variant TNF-alpha proteinscan be replaced by the same amino acid but of the opposite chirality.Thus, any amino acid naturally occurring in the L-configuration (whichmay also be referred to as the R or S, depending upon the structure ofthe chemical entity) may be replaced with an amino acid of the samechemical structural type, but of the opposite chirality, generallyreferred to as the D-amino acid but which can additionally be referredto as the R- or the S-, depending upon its composition and chemicalconfiguration. Such derivatives have the property of greatly increasedstability, and therefore are advantageous in the formulation ofcompounds which may have longer in vivo half lives, when administered byoral, intravenous, intramuscular, intraperitoneal, topical, rectal,intraocular, or other routes. In the preferred embodiment, the aminoacids are in the S- or L-configuration. If non-naturally occurring sidechains are used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Proteins including non-naturallyoccurring amino acids may be synthesized or in some cases, maderecombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22,1998 and Tang et al., Abstr. Pap Am. Chem. S218:U138-U138 Part 2 Aug.22, 1999, both of which are expressly incorporated by reference herein.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylainines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids may be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO.sub.3H) threonine, serine, tyrosine.

Other substitutions may include unnatural hydroxylated amino acids whichmay made by combining “alkyl” with any natural amino acid. The term“alkyl” as used herein refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Alkyl includesheteroalkyl, with atoms of nitrogen, oxygen and sulfur. Preferred alkylgroups herein contain 1 to 12 carbon atoms. Basic amino acids may besubstituted with alkyl groups at any position of the naturally occurringamino acids lysine, arginine, ornithine, citrulline, or(guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where“alkyl” is define as above. Nitrile derivatives (e.g., containing theCN-moiety in place of COOH) may also be substituted for asparagine orglutamine, and methionine sulfoxide may be substituted for methionine.Methods of preparation of such peptide derivatives are well known to oneskilled in the art.

In addition, any amide linkage in any of the variant TNF-alphapolypeptides can be replaced by a ketomethylene moiety. Such derivativesare expected to have the property of increased stability to degradationby enzymes, and therefore possess advantages for the formulation ofcompounds which may have increased in vivo half lives, as administeredby oral, intravenous, intramuscular, intraperitoneal, topical, rectal,intraocular, or other routes.

Additional amino acid modifications of amino acids of variant TNF-alphapolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues may also be derivatized by reaction with compounds such asbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such asimidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se is well-known, suchas for introducing spectral labels into tyrosyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidizoland tetranitromethane may be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

The TNF-alpha proteins may be from any number of organisms, withTNF-alpha proteins from mammals being particularly preferred. Suitablemammals include, but are not limited to, rodents (rats, mice, hamsters,guinea pigs, etc.), primates, farm animals (including sheep, goats,pigs, cows, horses, etc); and in the most preferred embodiment, fromhumans (the sequence of which is depicted in FIG. 6B). As will beappreciated by those in the art, TNF-alpha proteins based on TNF-alphaproteins from mammals other than humans may find use in animal models ofhuman disease.

The TNF proteins of the invention are antagonists of wild typeTNF-alpha. By “antagonists of wild type TNF-alpha” herein is meant thatthe variant TNF-alpha protein inhibits or significantly decreases theactivation of receptor signaling by wild type TNF-alpha proteins. In apreferred embodiment, the variant TNF-alpha protein interacts with thewild type TNF-alpha protein such that the complex comprising the variantTNF-alpha and wild type TNF-alpha is incapable of activating TNFreceptors, i.e. p55 TNF-R or p75 TNF-R.

In a preferred embodiment, the variant TNF-alpha protein is a variantTNF-alpha protein which functions as an antagonist of wild typeTNF-alpha. Preferably, the variant TNF-alpha protein preferentiallyinteracts with wild type TNF-alpha to form mixed trimers with the wildtype protein such that receptor binding does not occur and/or TNF-alphasignaling is not initiated (FIG. 1A).

By mixed trimers herein is meant that monomers of wild type and variantTNF-alpha proteins interact to form trimeric TNF-alpha (FIG. 5). Mixedtrimers may comprise 1 variant TNF-alpha protein:2 wild type TNF-alphaproteins, 2 variant TNF-alpha proteins:1 wild type TNF-alpha protein. Insome embodiments, trimers may be formed comprising only variantTNF-alpha proteins (FIG. 1B).

The variant TNF-alpha antagonist proteins of the invention are highlyspecific for TNF-alpha antagonism relative to TNF-beta antagonism.Additional characteristics include improved stability, pharmacokinetics,and high affinity for wild type TNF-alpha. Variants with higher affinitytoward wild type TNF-alpha may be generated from variants exhibitingTNF-alpha antagonism as outlined above.

In a preferred embodiment, variant TNF-alpha proteins exhibit decreasedbiological activity as compared to wild type TNF-alpha, including butnot limited to, decreased binding to the receptor, decreased activationand/or ultimately a loss of cytotoxic activity. By “cytotoxic activity”herein refers to the ability of a TNF-alpha variant to selectively killor inhibit cells. Variant TNF-alpha proteins that exhibit less than 50%biological activity as compared to wild type are preferred. Morepreferred are variant TNF-alpha proteins that exhibit less than 25%,even more preferred are variant proteins that exhibit less than 15%, andmost preferred are variant TNF-alpha proteins that exhibit less than 10%of a biological activity of wild-type TNF-alpha. Suitable assaysinclude, but are not limited to, TNF-alpha cytotoxicity assays, DNAbinding assays; transcription assays (using reporter constructs; seeStavridi, supra); size exclusion chromatography assays andradiolabeling/immuno-precipitation; see Corcoran et al., supra); andstability assays (including the use of circular dichroism (CD) assaysand equilibrium studies; see Mateu, supra); all of which are expresslyincorporated by reference.

In one embodiment, at least one property critical for binding affinityof the variant TNF-alpha proteins is altered when compared to the sameproperty of wild type TNF-alpha and in particular, variant TNF-alphaproteins with altered receptor affinity are preferred. Particularlypreferred are variant TNF-alpha with altered affinity towardoligomerization to wild type TNF-alpha.

Thus, the invention provides variant TNF-alpha proteins with alteredbinding affinities such that the variant TNF-alpha proteins willpreferentially oligomerize with wild type TNF-alpha, but do notsubstantially interact with wild type TNF receptors, i.e., p55, p75.“Preferentially” in this case means that given equal amounts of variantTNF-alpha monomers and wild type TNF-alpha monomers, at least 25% of theresulting trimers are mixed trimers of variant and wild type TNF-alpha,with at least about 50% being preferred, and at least about 80-90% beingparticularly preferred. In other words, it is preferable that thevariant TNF-alpha proteins of the invention have greater affinity forwild type TNF-alpha protein as compared to wild type TNF-alpha proteins.By “do not substantially interact with TNF receptors” herein is meantthat the variant TNF-alpha proteins will not be able to associate witheither the p55 or p75 receptors to activate the receptor and initiatethe TNF signaling pathway(s). In a preferred embodiment, at least a 50%decrease in receptor activation is seen, with greater than 50%, 76%,80-90% being preferred.

As outlined above, the invention provides variant TNF-alpha nucleicacids encoding variant TNF-alpha polypeptides. The variant TNF-alphapolypeptide preferably has at least one altered property as compared tothe same property of the corresponding naturally occurring TNFpolypeptide. The property of the variant TNF-alpha polypeptide is theresult the PDA analysis of the present invention.

The term “altered property” or grammatical equivalents thereof in thecontext of a polypeptide, as used herein, further refers to anycharacteristic or attribute of a polypeptide that can be selected ordetected and compared to the corresponding property of a naturallyoccurring protein. These properties include, but are not limited tocytotoxic activity; oxidative stability, substrate specificity,substrate binding or catalytic activity, thermal stability, alkalinestability, pH activity profile, resistance to proteolytic degradation,kinetic association (Kon) and dissociation (Koff) rate, protein folding,inducing an immune response, ability to bind to a ligand, ability tobind to a receptor, ability to be secreted, ability to be displayed onthe surface of a cell, ability to oligomerize, ability to signal,ability to stimulate cell proliferation, ability to inhibit cellproliferation, ability to induce apoptosis, ability to be modified byphosphorylation or glycosylation, and the ability to treat disease.

Unless otherwise specified, a substantial change in any of theabove-listed properties, when comparing the property of a variantTNF-alpha polypeptide to the property of a naturally occurring TNFprotein is preferably at least a 20%, more preferably, 50%, morepreferably at least a 2-fold increase or decrease. A change in cytotoxicactivity is evidenced by at least a 75% or greater decrease in celldeath initiated by a variant TNF-alpha protein as compared to wild typeprotein.

A change in binding affinity is evidenced by at least a 5% or greaterincrease or decrease in binding affinity to wild type TNF receptorproteins or to wild type TNF-alpha.

A change in oxidative stability is evidenced by at least about 20%, morepreferably at least 50% increase of activity of a variant TNF-alphaprotein when exposed to various oxidizing conditions as compared to thatof wild type TNF-alpha. Oxidative stability is measured by knownprocedures.

A change in alkaline stability is evidenced by at least about a 5% orgreater increase or decrease (preferably increase) in the half-life ofthe activity of a variant TNF-alpha protein when exposed to increasingor decreasing pH conditions as compared to that of wild type TNF-alpha.Generally, alkaline stability is measured by known procedures.

A change in thermal stability is evidenced by at least about a 5% orgreater increase or decrease (preferably increase) in the half-life ofthe activity of a variant TNF-alpha protein when exposed to a relativelyhigh temperature and neutral pH as compared to that of wild typeTNF-alpha. Generally, thermal stability is measured by known procedures.

Similarly, variant TNF-alpha proteins, for example are experimentallytested and validated in in vivo and in in vitro assays. Suitable assaysinclude, but are not limited to, activity assays and binding assays. Forexample, TNF-alpha activity assays, such as detecting apoptosis viacaspase activity can be used to screen for TNF-alpha variants that areantagonists of wild type TNF-alpha. Other assays include using the Sytoxgreen nucleic acid stain to detect TNF-induced cell permeability in anActinomycin-D sensitized cell line. As this stain is excluded from livecells, but penetrates dying cells, this assay also can be used to detectTNF-alpha variants that are agonists of wild-type TNF-alpha. By“agonists of “wild type TNF-alpha” herein is meant that the variantTNF-alpha protein enhances the activation of receptor signaling by wildtype TNF-alpha proteins. Generally, variant TNF-alpha proteins thatfunction as agonists of wild type TNF-alpha are not preferred. However,in some embodiments, variant TNF-alpha proteins that function asagonists of wild type TNF-alpha protein are preferred. An example of anNF kappaB assay is presented in Example 7.

In a preferred embodiment, binding affinities of variant TNF-alphaproteins as compared to wild type TNF-alpha proteins for naturallyoccurring TNF-alpha and TNF receptor proteins such as p55 and p75 aredetermined. Suitable assays include, but are not limited to, e.g.,quantitative comparisons comparing kinetic and equilibrium bindingconstants. The kinetic association rate (Kon) and dissociation rate(Koff), and the equilibrium binding constants (Kd) may be determinedusing surface plasmon resonance on a BIAcore instrument following thestandard procedure in the literature [Pearce et al., Biochemistry38:81-89 (1999)]. Again, as outlined herein, variant TNF-alpha proteinsthat preferentially form mixed trimers with wild type TNF-alphaproteins, but do not substantially interact with wild type receptorproteins are preferred. Examples of binding assays are described inExample 6.

In a preferred embodiment, the antigenic profile in the host animal ofthe variant TNF-alpha protein is similar, and preferably identical, tothe antigenic profile of the host TNF-alpha; that is, the variantTNF-alpha protein does not significantly stimulate the host organism(e.g. the patient) to an immune response; that is, any immune responseis not clinically relevant and there is no allergic response orneutralization of the protein by an antibody. That is, in a preferredembodiment, the variant TNF-α protein does not contain additional ordifferent epitopes from the TNF-alpha. By “epitope” or “determinant”herein is meant a portion of a protein which will generate and/or bindan antibody. Thus, in most instances, no significant amounts ofantibodies are generated to a variant TNF-alpha protein. In general,this is accomplished by not significantly altering surface residues, asoutlined below nor by adding any amino acid residues on the surfacewhich can become glycosylated, as novel glycosylation can result in animmune response. The variant TNF-alpha proteins and nucleic acids of theinvention are distinguishable from naturally occurring wild typeTNF-alpha. By “naturally occurring” or “wild type” or grammaticalequivalents, herein is meant an amino acid sequence or a nucleotidesequence that is found in nature and includes allelic variations; thatis, an amino acid sequence or a nucleotide sequence that usually has notbeen intentionally modified. Accordingly, by “non-naturally occurring”or “synthetic” or “recombinant” or grammatical equivalents thereof,herein is meant an amino acid sequence or a nucleotide sequence that isnot found in nature; that is, an amino acid sequence or a nucleotidesequence that usually has been intentionally modified. It is understoodthat once a recombinant nucleic acid is made and reintroduced into ahost cell or organism, it will replicate non-recombinantly, i.e., usingthe in vivo cellular machinery of the host cell rather than in vitromanipulations, however, such nucleic acids, once produced recombinantly,although subsequently replicated non-recombinantly, are still consideredrecombinant for the purpose of the invention. Representative amino acidand nucleotide sequences of a naturally occurring human TNF-alpha areshown in FIG. 6A and 6B (SEQ ID NOS:1-2). It should be noted, thatunless otherwise stated, all positional numbering of variant TNT-alphaproteins and variant TNF-alpha nucleic acids is based on thesesequences. That is, as will be appreciated by those in the art, analignment of TNF-alpha proteins and variant TNT-alpha proteins may bedone using standard programs, as is outlined below, with theidentification of “equivalent” positions between the two proteins. Thus,the variant TNT-alpha proteins and nucleic acids of the invention arenon-naturally occurring; that is, they do not exist in nature.

Thus, in a preferred embodiment, the variant TNF-alpha protein has anamino acid sequence that differs from a wild type TNF-alpha sequence byat least 1-5% of the residues. That is, the variant TNF-alpha proteinsof the invention are less than about 97-99% identical to a wild typeTNF-alpha amino acid sequence. Accordingly, a protein is a “variantTNF-alpha protein” if the overall homology of the protein sequence tothe amino acid sequence shown in FIG. 6 is preferably less than about99%, more preferably less than about 98%, even more preferably less thanabout 97% and more preferably less than 95%. In some embodiments, thehomology will be as low as about 75-80%. Stated differently, based onthe human TNF sequence of FIG. 6B, variant TNF-alpha proteins have atleast about 1 residue that differs from the human TNF-alpha sequence,with at least about 2, 3, 4, or 5 different residues. Preferred variantTNF-alpha proteins have 3 to 5 different residues.

Homology in this context means sequence similarity or identity, withidentity being preferred. As is known in the art, a number of differentprograms may be used to identify whether a protein (or nucleic acid asdiscussed below) has sequence identity or similarity to a knownsequence. Sequence identity and/or similarity is determined usingstandard techniques known in the art, including, but not limited to, thelocal sequence identity algorithm of Smith & Waterman, Adv. Appl. Math.,2:482 (1981), by the sequence identity alignment algorithm of Needleman& Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similaritymethod of Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fitsequence program described by Devereux et al., Nucl. Acid Res.,12:387-395 (1984), preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pair wise alignments. It may also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul etal., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Methods in Enzymology, 266:460-480 (1996)]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST, as reported by Altschulet al., Nucl. Acids Res., 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by ascore corresponding to ˜22 bits.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the coding sequenceof the cell cycle protein. A preferred method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequence of FIG. 6B,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than that shown in FIG. 6, asdiscussed below, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitymay be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Thus, the variant TNF-alpha proteins of the present invention may beshorter or longer than the amino acid sequence shown in FIG. 6B (SEQ IDNO:2). As used in this invention, “wild type TNF-alpha” is a nativemammalian protein (preferably human). TNF-alpha is polymorphic. Anexample of the amino acid sequences shown in FIG. 6B. Thus, in apreferred embodiment, included within the definition of variant TNFproteins are portions or fragments of the sequences depicted herein.Fragments of variant TNF-alpha proteins are considered variant TNF-alphaproteins if a) they share at least one antigenic epitope; b) have atleast the indicated homology; c) and preferably have variant TNF-alphabiological activity as defined herein.

In a preferred embodiment, as is more fully outlined below, the variantTNF-alpha proteins include further amino acid variations, as compared toa wild type TNF-alpha, than those outlined herein. In addition, asoutlined herein, any of the variations depicted herein may be combinedin any way to form additional novel variant TNF-alpha proteins.

In addition, variant TNF-alpha proteins may be made that are longer thanthose depicted in the figures, for example, by the addition of epitopeor purification tags, as outlined herein, the addition of other fusionsequences, etc. For example, the variant TNF-alpha proteins of theinvention may be fused to other therapeutic proteins or to otherproteins such as Fc or serum albumin for pharmacokinetic purposes. Seefor example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which areexpressly incorporated by reference.

In a preferred embodiment, the variant TNF-alpha proteins compriseresidues selected from the following positions 21, 23, 30, 31, 32, 33,34, 35, 57, 65, 66, 67, 75, 84, 86, 87, 91, 97, 111, 112, 115, 140, 143,144, 145, 146, and 147.

Also included within the invention are variant TNF-alpha proteinscomprising variable residues in core, surface, and boundary residues.

Preferred amino acids for each position, including the human TNF-alpharesidues, are shown in FIG. 7 (SEQ ID NO:3-24). Thus, for example, atposition 143, preferred amino acids are Glu, Asn, Gln, Ser, Arg, andLys; etc.

Preferred changes include: Q21C, Q21R, E23C, N34E, V91E, Q21R, N30D,R31C, R31I, R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C,L57F, L57W, L57Y, K65D, K65E, K65I, K65M, K65N, K65Q, K65T, K65S, K65V,K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, L75E, L75K, L75Q,A84V, S86Q, S86R, Y87H, Y87R, V91E, I97R, I97T, A111R, A111E, K112D,K112E, Y115D, Y115E, Y115F, Y115H, Y115I, Y115K, Y115L, Y115M, Y115N,Y115Q, Y115R, Y115S, Y115T, Y115W, D140K, D140R, D143E, D143K, D143L,D143R, D143N, D143Q, D143R, D143S, F144N, A145D, A145E, A145F, A145H,A145K, A145M, A145N, A145Q, A145R, A145S, A145T, A145Y, E146K, E146L,E146M, E146N, E146R, E146S and S147R.

These may be done either individually or in combination, with anycombination being possible. However, as outlined herein, preferredembodiments utilize at least 1 to 5, and preferably more, positions ineach variant TNF-alpha protein.

For purposes of the present invention, the areas of the wild type ornaturally occurring TNF-alpha molecule to be modified are selected fromthe group consisting of the Large Domain (also known as II), SmallDomain (also known as I), the DE loop, and the trimer interface. TheLarge Domain, the Small Domain and the DE loop are the receptorinteraction domains. The modifications may be made solely in one ofthese areas or in any combination of these areas.

The Large Domain preferred positions to be varied include: 21, 30, 31,32, 33, 35, 65, 66, 67, 111, 112, 115, 140, 143, 144, 145, 146 and/or147 (FIG. 11). For the Small Domain, the preferred positions to bemodified are 75 and/or 97. For the DE Loop, the preferred positionmodifications are 84, 86, 87 and/or 91. The Trimer Interface haspreferred double variants including positions 34 and 91 as well as atposition 57.

In a preferred embodiment, substitutions at multiple receptorinteraction and/or trimerization domains may be combined. Examplesinclude, but are not limited to, simultaneous substitution of aminoacids at the large and small domains (e.g. A145R and I97T), large domainand DE loop (A145R and Y87H), and large domain and trimerization domain(A145R and L57F). Additional examples include any and all combinations,e.g., I97T and Y87H (small domain and DE loop).

More specifically, theses variants may be in the form of single pointvariants, for example K112D, Y115K, Y115I, Y115T, A145E or A145R. Thesesingle point variants may be combined, for example, Y115I and A145E, orY115I and A145R, or Y115T and A145R or Y115I and A145E; or any othercombination.

Preferred double point variant positions include 57, 75, 86, 87, 97,115, 143, 145, and 146; in any combination.

In addition, double point variants may be generated including L57F andone of Y115I, Y115Q, Y115T, D143K, D143R, D143E, A145E, A145R, E146K orE146R.

Other preferred double variants are Y115Q and at least one of D143N,D143Q, A145K, A145R, or E146K; Y115M and at least one of D143N, D143Q,A145K, A145R or E146K; and L57F and at least one of A145E or 146R; K65Dand either D143K or D143R, K65E and either D143K or D143R, Y115Q and anyof L75Q, L57W, L57Y, L57F, I97R, I97T, S86Q, D143N, E146K, A145R andI97T, A145R and either Y87R or Y87H; N34E and V91E; L75E and Y115Q; L75Qand Y115Q; L75E and A145R; and L75Q and A145R.

Further, triple point variants may be generated. Preferred positionsinclude 34, 75, 87, 91, 115, 143, 145 and 146. Examples of triple pointvariants include V91E, N34E and one of Y115I, Y115T, D143K, D143R,A145R, A145E E146K, and E146R. Other triple point variants include L75Eand Y87H and at least one of Y115Q, A145R, Also, L75K, Y87H and Y115Q.More preferred are the triple point variants V91E, N34E and either A145Ror A145E.

In a preferred embodiment, the variant TNF-alpha proteins of theinvention are human TNF-alpha conformers. By “conformer” herein is meanta protein that has a protein backbone 3-D structure that is virtuallythe same but has significant differences in the amino acid side chains.That is, the variant TNF-alpha proteins of the invention define aconformer set, wherein all of the proteins of the set share a backbonestructure and yet have sequences that differ by at least 1-3-5%. Thethree dimensional backbone structure of a variant TNF-alpha protein thussubstantially corresponds to the three-dimensional backbone structure ofhuman TNF-alpha. “Backbone” in this context means the non-side chainatoms: the nitrogen, carbonyl carbon and oxygen, and the α-carbon, andthe hydrogens attached to the nitrogen and α-carbon. To be considered aconformer, a protein must have backbone atoms that are no more than 2Angstroms RMSD from the human TNF-alpha structure, with no more than 1.5Angstroms RMSD being preferred, and no more than 1 Angstrom RMSD beingparticularly preferred. In general, these distances may be determined intwo ways. In one embodiment, each potential conformer is crystallizedand its three-dimensional structure determined. Alternatively, as theformer is quite tedious, the sequence of each potential conformer is runin the PDATM technology program to determine whether it is a conformer.

Variant TNF-alpha proteins may also be identified as being encoded byvariant TNF-alpha nucleic acids. In the case of the nucleic acid, theoverall homology of the nucleic acid sequence is commensurate with aminoacid homology but takes into account the degeneracy in the genetic codeand codon bias of different organisms. Accordingly, the nucleic acidsequence homology may be either lower or higher than that of the proteinsequence, with lower homology being preferred.

In a preferred embodiment, a variant TNF-alpha nucleic acid encodes avariant TNF-alpha protein. As will be appreciated by those in the art,due to the degeneracy of the genetic code, an extremely large number ofnucleic acids may be made, all of which encode the variant TNF-alphaproteins of the present invention. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids, by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of thevariant TNF-alpha.

In one embodiment, the nucleic acid homology is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequence shown in FIG. 6A (SEQID NO:1) or its complement and encode a variant TNF-alpha protein isconsidered a variant TNF-alpha gene.

High stringency conditions are known in the art; see for exampleManiatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition,1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al.,both of which are hereby incorporated by reference. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10 degrees C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionicstrength, pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30 degreesC. for short probes (e.g. 10 to 50 nucleotides) and at least about 60degrees C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

The variant TNF-alpha proteins and nucleic acids of the presentinvention are recombinant. As used herein, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half-life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequence depicted in FIG. 6 also includes the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated variant TNF-alpha nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in itswild-type host, and thus may be substantially pure. For example, anisolated protein is unaccompanied by at least some of the material withwhich it is normally associated in its natural state, preferablyconstituting at least about 0.5%, more preferably at least about 5% byweight of the total protein in a given sample. A substantially pureprotein comprises at least about 75% by weight of the total protein,with at least about 80% being preferred, and at least about 90% beingparticularly preferred. The definition includes the production of avariant TNF-alpha protein from one organism in a different organism orhost cell. Alternatively, the protein may be made at a significantlyhigher concentration than is normally seen, through the use of ainducible promoter or high expression promoter, such that the protein ismade at increased concentration levels. Furthermore, all of the variantTNF-alpha proteins outlined herein are in a form not normally found innature, as they contain amino acid substitutions, insertions anddeletions, with substitutions being preferred, as discussed below.

Also included within the definition of variant TNF-alpha proteins of thepresent invention are amino acid sequence variants of the variantTNF-alpha sequences outlined herein and shown in the Figures. That is,the variant TNF-alpha proteins may contain additional variable positionsas compared to human TNF-alpha. These variants fall into one or more ofthree classes: substitutional, insertional or deletional variants. Thesevariants ordinarily are prepared by site-specific mutagenesis ofnucleotides in the DNA encoding a variant TNF-alpha protein, usingcassette or PCR mutagenesis or other techniques well known in the art,to produce DNA encoding the variant, and thereafter expressing the DNAin recombinant cell culture as outlined above. However, variantTNF-alpha protein fragments having up to about 100-150 residues may beprepared by in vitro synthesis using established techniques. Amino acidsequence variants are characterized by the predetermined nature of thevariation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the variant TNF-alpha protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue; althoughvariants can also be selected which have modified characteristics aswill be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variant TNF-alpha proteinsscreened for the optimal combination of desired activity. Techniques formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, M13 primer mutagenesis andPCR mutagenesis. Screening of the mutants is done using assays ofvariant TNF-alpha protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the variant TNF-alphaprotein are desired, substitutions are generally made in accordance withthe following chart:

CHART 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, TyrSer Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the original variantTNF-alpha protein, although variants also are selected to modify thecharacteristics of the variant TNF-alpha proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the variant TNF-alpha protein is altered. For example,glycosylation sites may be altered or removed. Similarly, the biologicalfunction may be altered; for example, in some instances it may bedesirable to have more or less potent TNF-alpha activity.

In a preferred embodiment, also included within the invention aresoluble p55 variant TNF proteins and nucleic acids. In this embodiment,the soluble p55 variant TNF can serve as an antagonist to receptorsignaling. By “serving as an antagonist to receptor signaling” herein ismeant that the soluble p55 variant TNF proteins preferentially interactwith wild type TNF-alpha to block or significantly decrease TNF-alphareceptor activated signaling.

Thus, the computational processing results described above may be usedto generate a set of optimized variant p55 protein sequences. Optimizedvariant p55 protein sequences are generally different from wild type p55sequences in at least about 1 variant amino acid.

In a preferred embodiment variant TNF p55 proteins are fused to a humanTNF receptor-associated factor (TRAF) trimerization domain (FIG. 12). Ina preferred embodiment, the C termini of optimized variant TNF p 55receptors will be fused to TRAF trimerization domains (i.e., leucinezipper motif). Fusion of trimerization domains from TRAF proteins toTNFR molecules can induce trimerization, resulting in higher avidity forTNF-alpha thereby creating a more potent TNF-alpha inhibitor than themonomeric soluble TNFR. These trimerization domains can be used toinduce the trimerization of any protein where this may be desirable,including TNF-alpha, TNF beta, TNF receptor (p55 and p75), and othermembers of the TNF receptor family including NGF receptor, CD27, CD30,CD40, fas antigen. Other peptides that are known to form trimeric coiledcoils could also be used, including pII (Harbury, Kim and Alber, 1994).

While the description herein is focused on TNF-alpha variants, as willbe appreciated by those in the art, the embodiments and definitions canbe applied to soluble p55 variant TNF proteins.

The variant TNF-alpha proteins and nucleic acids of the invention can bemade in a number of ways. Individual nucleic acids and proteins can bemade as known in the art and outlined below. Alternatively, libraries ofvariant TNF-alpha proteins can be made for testing.

In a preferred embodiment, sets or libraries of variant TNF-alphaproteins are generated from a probability distribution table. Asoutlined herein, there are a variety of methods of generating aprobability distribution table, including using PDATM technologycalculations, sequence alignments, forcefield calculations such as SCMFcalculations, etc. In addition, the probability distribution can be usedto generate information entropy scores for each position, as a measureof the mutational frequency observed in the library.

In this embodiment, the frequency of each amino acid residue at eachvariable position in the list is identified. Frequencies may bethresholded, wherein any variant frequency lower than a cutoff is set tozero. This cutoff is preferably 1%, 2%, 5%, 10% or 20%, with 10% beingparticularly preferred. These frequencies are then built into thevariant TNF-alpha library. That is, as above, these variable positionsare collected and all possible combinations are generated, but the aminoacid residues that “fill” the library are utilized on a frequency basis.Thus, in a non-frequency based library, a variable position that has 5possible residues will have 20% of the proteins comprising that variableposition with the first possible residue, 20% with the second, etc.However, in a frequency based library, a variable position that has 5possible residues with frequencies of 10%, 15%, 25%, 30% and 20%,respectively, will have 10% of the proteins comprising that variableposition with the first possible residue, 15% of the proteins with thesecond residue, 25% with the third, etc. As will be appreciated by thosein the art, the actual frequency may depend on the method used toactually generate the proteins; for example, exact frequencies may bepossible when the proteins are synthesized. However, when thefrequency-based primer system outlined below is used, the actualfrequencies at each position will vary, as outlined below.

As will be appreciated by those in the art and outlined herein,probability distribution tables can be generated in a variety of ways.In addition to the methods outlined herein, self-consistent mean field(SCMF) methods can be used in the direct generation of probabilitytables. SCMF is a deterministic computational method that uses a meanfield description of rotamer interactions to calculate energies. Aprobability table generated in this way can be used to create librariesas described herein. SCMF can be used in three ways: the frequencies ofamino acids and rotamers for each amino acid are listed at eachposition; the probabilities are determined directly from SCMF (seeDelarue et la. Pac. Symp. Biocomput. 109-21 (1997), expresslyincorporated by reference). In addition, highly variable positions andnon-variable positions can be identified. Alternatively, another methodis used to determine what sequence is jumped to during a search ofsequence space; SCMF is used to obtain an accurate energy for thatsequence; this energy is then used to rank it and create a rank-orderedlist of sequences (similar to a Monte Carlo sequence list). Aprobability table showing the frequencies of amino acids at eachposition can then be calculated from this list (Koehl et al., J. Mol.Biol. 239:249 (1994); Koehl et al., Nat. Struc. Biol. 2:163 (1995);Koehl et al., Curr. Opin. Struct. Biol. 6:222 (1996); Koehl et al., J.Mol. Bio. 293:1183 (1999); Koehl et al., J. Mol. Biol. 293:1161 (1999);Lee J. Mol. Biol. 236:918 (1994); and Vasquez Biopolymers 36:53-70(1995); all of which are expressly incorporated by reference. Similarmethods include, but are not limited to, OPLS-AA (Jorgensen, et al., J.Am. Chem. Soc. (1996), v 118, pp 11225-11236; Jorgensen, W. L.; BOSS,Version 4.1; Yale University: New Haven, Conn. (1999)); OPLS (Jorgensen,et al., J. Am. Chem. Soc. (1988), v 110, pp 1657ff; Jorgensen, et al.,J. Am. Chem. Soc. (1990), v 112, pp 4768ff); UNRES (United ResidueForcefield; Liwo, et al., Protein Science (1993), v 2, pp1697-1714;Liwo, et al., Protein Science (1993), v 2, pp1715-1731; Liwo, et al., J.Comp. Chem. (1997), v 18, pp849-873; Liwo, et al., J. Comp. Chem.(1997), v 18, pp874-884; Liwo, et al., J. Comp. Chem. (1998), v 19,pp259-276; Forcefield for Protein Structure Prediction (Liwo, et al.,Proc. Natl. Acad. Sci. USA (1999), v 96, pp5482-5485); ECEPP/3 (Liwo etal., J Protein Chem 1994 May; 13(4):375-80); AMBER 1.1 force field(Weiner, et al., J. Am. Chem. Soc. v106, pp765-784); AMBER 3.0 forcefield (U. C. Singh et al., Proc. Natl. Acad. Sci. USA. 82:755-759);CHARMM and CHARMM22 (Brooks, et al., J. Comp. Chem. v4, pp 187-217);cvff3.0 (Dauber-Osguthorpe, et al., (1988) Proteins: Structure, Functionand Genetics, v4,pp31-47); cff91 (Maple, et al., J. Comp. Chem. v15,162-182); also, the DISCOVER (cvff and cff91) and AMBER forcefields areused in the INSIGHT molecular modeling package (Biosym/MSI, San DiegoCalif.) and HARMM is used in the QUANTA molecular modeling package(Biosym/MSI, San Diego Calif.).

In addition, as outlined herein, a preferred method of generating aprobability distribution table is through the use of sequence alignmentprograms. In addition, the probability table may be obtained by acombination of sequence alignments and computational approaches. Forexample, one may add amino acids found in the alignment of homologoussequences to the result of the computation. Preferable one may add thewild-type amino acid identity to the probability table if it is notfound in the computation.

In an alternative embodiment, TNF-alpha variants are designed using thecomputational techniques described above. In this alternativeembodiment, non-naturally occurring TNF-alpha monomer or dimer variantsare generated to bind to the receptor. More preferably, these variantspreferably bind to the receptor and competitively inhibit naturallyoccurring TNF-alpha molecules to bind to the receptor. The dimervariants are more preferred as they substantially bind to the receptorinterface.

Preferred examples of these variants are modified at the followingpositions: 6, 7, 8, 9, 10, 11, 13, 15, 33, 34, 36, 53, 54, 55, 57, 59,61, 63, 69, 72, 73, 75, 82, 87, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 106, 107, 109, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 146, 147, 148, 149, 151, 155, 156,and 157, and any and all combinations of these positions.

As will be appreciated, a variant TNF-alpha library created byrecombining variable positions and/or residues at the variable positionmay not be in a rank-ordered or filtered list. In some embodiments, theentire list may just be made and tested. Alternatively, in a preferredembodiment, the variant TNF-alpha library is also in the form of arank-ordered or filtered list. This may be done for several reasons,including the size of the library is still too big to generateexperimentally, or for predictive purposes. This may be done in severalways. In one embodiment, the library is ranked using the scoringfunctions of the PDA™ technology to rank the library members.Alternatively, statistical methods may be used. For example, the librarymay be ranked by frequency score; that is, proteins containing the mostof high frequency residues could be ranked higher, etc. This may be doneby adding or multiplying the frequency at each variable position togenerate a numerical score. Similarly, the library different positionsmay be weighted and then the proteins scored; for example, thosecontaining certain residues could be arbitrarily ranked.

In a preferred embodiment, the different protein members of the variantTNF-alpha library may be chemically synthesized. This is particularlyuseful when the designed proteins are short, preferably less than 150amino acids in length, with less than 100 amino acids being preferred,and less than 50 amino acids being particularly preferred, although asis known in the art, longer proteins may be made chemically orenzymatically. See for example Wilken et al, Curr. Opin. Biotechnol.9:412-26 (1998), hereby expressly incorporated by reference.

In a preferred embodiment, particularly for longer proteins or proteinsfor which large samples are desired, the library sequences are used tocreate nucleic acids such as DNA which encode the member sequences andwhich may then be cloned into host cells, expressed and assayed, ifdesired. Thus, nucleic acids, and particularly DNA, may be made whichencodes each member protein sequence. This is done using well knownprocedures. The choice of codons, suitable expression vectors andsuitable host cells will vary depending on a number of factors, and maybe easily optimized as needed.

In a preferred embodiment, multiple PCR reactions with pooledoligonucleotides are done, as is generally depicted in the FIGS. 13-17.In this embodiment, overlapping oligonucleotides are synthesized whichcorrespond to the full-length gene. Again, these oligonucleotides mayrepresent all of the different amino acids at each variant position orsubsets.

In a preferred embodiment, these oligonucleotides are pooled in equalproportions and multiple PCR reactions are performed to createfull-length sequences containing the combinations of mutations definedby the library. In addition, this may be done using error-prone PCRmethods.

In a preferred embodiment, the different oligonucleotides are added inrelative amounts corresponding to the probability distribution table.The multiple PCR reactions thus result in full length sequences with thedesired combinations of mutations in the desired proportions.

The total number of oligonucleotides needed is a function of the numberof positions being mutated and the number of mutations being consideredat these positions:

-   (number of oligos for constant positions)+M1+M2+M3+M_(n)=(total    number of oligos required), where Mn is the number of mutations    considered at position n in the sequence.

In a preferred embodiment, each overlapping oligonucleotide comprisesonly one position to be varied; in alternate embodiments, the variantpositions are too close together to allow this and multiple variants peroligonucleotide are used to allow complete recombination of all thepossibilities. That is, each oligo may contain the codon for a singleposition being mutated, or for more than one position being mutated. Themultiple positions being mutated must be close in sequence to preventthe oligo length from being impractical. For multiple mutating positionson an oligonucleotide, particular combinations of mutations may beincluded or excluded in the library by including or excluding theoligonucleotide encoding that combination. For example, as discussedherein, there may be correlations between variable regions; that is,when position X is a certain residue, position Y must (or must not) be aparticular residue. These sets of variable positions are sometimesreferred to herein as a “cluster”. When the clusters are comprised ofresidues close together, and thus can reside on one oligonucleotideprimer, the clusters can be set to the “good” correlations, andeliminate the bad combinations that may decrease the effectiveness ofthe library. However, if the residues of the cluster are far apart insequence, and thus will reside on different oligonucleotides forsynthesis, it may be desirable to either set the residues to the “good”correlation, or eliminate them as variable residues entirely. In analternative embodiment, the library may be generated in several steps,so that the cluster mutations only appear together. This procedure, i.e.the procedure of identifying mutation clusters and either placing themon the same oligonucleotides or eliminating them from the library orlibrary generation in several steps preserving clusters, canconsiderably enrich the experimental library with properly foldedprotein. Identification of clusters may be carried out by a number ofways, e.g. by using known pattern recognition methods, comparisons offrequencies of occurrence of mutations or by using energy analysis ofthe sequences to be experimentally generated (for example, if the energyof interaction is high, the positions are correlated). Thesecorrelations may be positional correlations (e.g. variable positions 1and 2 always change together or never change together) or sequencecorrelations (e.g. if there is residue A at position 1, there is alwaysresidue B at position 2). See: Pattern discovery in Biomolecular Data:Tools, Techniques, and Applications; edited by Jason T. L. Wang, BruceA. Shapiro, Dennis Shasha. New York: Oxford University, 1999; Andrews,Harry C. Introduction to mathematical techniques in pattern recognition;New York, Wiley-Interscience [1972]; Applications of PatternRecognition; Editor, K. S. Fu. Boca Raton, Fla. CRC Press, 1982; GeneticAlgorithms for Pattern Recognition; edited by Sankar K. Pal, Paul P.Wang. Boca Raton: CRC Press, c1996; Pandya, Abhijit S., Patternrecognition with neural networks in C++/Abhijit S. Pandya, Robert B.Macy. Boca Raton, Fla.: CRC Press, 1996; Handbook of pattern recognition& computer vision/edited by C. H. Chen, L. F. Pau, P. S. P. Wang. 2nded. Singapore; River Edge, N.J.: World Scientific, c1999; Friedman,Introduction to Pattern Recognition: Statistical, Structural, Neural,and Fuzzy Logic Approaches; River Edge, N.J.: World Scientific, c1999,Series title: Series in machine perception and artificial intelligence;vol. 32; all of which are expressly incorporated by reference. Inaddition, programs used to search for consensus motifs can be used aswell.

In addition, correlations and shuffling can be fixed or optimized byaltering the design of the oligonucleotides; that is, by deciding wherethe oligonucleotides (primers) start and stop (e.g. where the sequencesare “cut”). The start and stop sites of oligos can be set to maximizethe number of clusters that appear in single oligonucleotides, therebyenriching the library with higher scoring sequences. Differentoligonucleotide start and stop site options can be computationallymodeled and ranked according to number of clusters that are representedon single oligos, or the percentage of the resulting sequencesconsistent with the predicted library of sequences.

The total number of oligonucleotides required increases when multiplemutable positions are encoded by a single oligonucleotide. The annealedregions are the ones that remain constant, i.e. have the sequence of thereference sequence.

Oligonucleotides with insertions or deletions of codons may be used tocreate a library expressing different length proteins. In particularcomputational sequence screening for insertions or deletions may resultin secondary libraries defining different length proteins, which can beexpressed by a library of pooled oligonucleotide of different lengths.

In a preferred embodiment, the variant TNF-alpha library is done byshuffling the family (e.g. a set of variants); that is, some set of thetop sequences (if a rank-ordered list is used) can be shuffled, eitherwith or without error-prone PCR. “Shuffling” in this context means arecombination of related sequences, generally in a random way. It caninclude “shuffling” as defined and exemplified in U.S. Pat. Nos.5,830,721; 5,811,238; 5,605,793; 5,837,458 and PCT US/19256, all ofwhich are expressly incorporated by reference in their entirety. Thisset of sequences may also be an artificial set; for example, from aprobability table (for example generated using SCMF) or a Monte Carloset. Similarly, the “family” can be the top 10 and the bottom 10sequences, the top 100 sequence, etc. This may also be done usingerror-prone PCR.

Thus, in a preferred embodiment, in silico shuffling is done using thecomputational methods described herein. That is, starting with eithertwo libraries or two sequences, random recombinations of the sequencesmay be generated and evaluated.

In a preferred embodiment, error-prone PCR is done to generate thevariant TNF-alpha library. See U.S. Pat. Nos. 5,605,793, 5,811,238, and5,830,721, all of which are hereby incorporated by reference. This maybe done on the optimal sequence or on top members of the library, orsome other artificial set or family. In this embodiment, the gene forthe optimal sequence found in the computational screen of the primarylibrary may be synthesized. Error-prone PCR is then performed on theoptimal sequence gene in the presence of oligonucleotides that code forthe mutations at the variant positions of the library (biasoligonucleotides). The addition of the oligonucleotides will create abias favoring the incorporation of the mutations in the library.Alternatively, only oligonucleotides for certain mutations may be usedto bias the library.

In a preferred embodiment, gene shuffling with error-prone PCR can beperformed on the gene for the optimal sequence, in the presence of biasoligonucleotides, to create a DNA sequence library that reflects theproportion of the mutations found in the variant TNF-alpha library. Thechoice of the bias oligonucleotides can be done in a variety of ways;they can chosen on the basis of their frequency, i.e. oligonucleotidesencoding high mutational frequency positions can be used; alternatively,oligonucleotides containing the most variable positions can be used,such that the diversity is increased; if the secondary library isranked, some number of top scoring positions may be used to generatebias oligonucleotides; random positions may be chosen; a few top scoringand a few low scoring ones may be chosen; etc. What is important is togenerate new sequences based on preferred variable positions andsequences.

In a preferred embodiment, PCR using a wild-type gene or other gene maybe used, as is schematically depicted in the Figures. In thisembodiment, a starting gene is used; generally, although this is notrequired, the gene is usually the wild-type gene. In some cases it maybe the gene encoding the global optimized sequence, or any othersequence of the list, or a consensus sequence obtained e.g. fromaligning homologous sequences from different organisms. In thisembodiment, oligonucleotides are used that correspond to the variantpositions and contain the different amino acids of the library. PCR isdone using PCR primers at the termini, as is known in the art. Thisprovides two benefits. First, this generally requires feweroligonucleotides and may result in fewer errors. Second, it hasexperimental advantages in that if the wild-type gene is used, it neednot be synthesized.

In addition, there are several other techniques that may be used, asexemplified in FIGS. 13-17. In a preferred embodiment, ligation of PCRproducts is done.

In a preferred embodiment, a variety of additional steps may be done tothe variant TNF-alpha library; for example, further computationalprocessing may occur, different variant TNF-alpha libraries can berecombined, or cutoffs from different libraries may be combined. In apreferred embodiment, a variant TNF-alpha library may be computationallyremanipulated to form an additional variant TNF-alpha library (sometimesreferred to herein as “tertiary libraries”). For example, any of thevariant TNF-alpha library sequences may be chosen for a second round ofPDA, by freezing or fixing some or all of the changed positions in thefirst library. Alternatively, only changes seen in the last probabilitydistribution table are allowed. Alternatively, the stringency of theprobability table may be altered, either by increasing or decreasing thecutoff for inclusion. Similarly, the variant TNF-alpha library may berecombined experimentally after the first round; for example, the bestgene/genes from the first screen may be taken and gene assembly redone(using techniques outlined below, multiple PCR, error-prone PCR,shuffling, etc.). Alternatively, the fragments from one or more goodgene(s) to change probabilities at some positions. This biases thesearch to an area of sequence space found in the first round ofcomputational and experimental screening.

In a preferred embodiment, a tertiary library may be generated fromcombining different variant TNF-alpha libraries. For example, aprobability distribution table from a first variant TNF-alpha librarymay be generated and recombined, either computationally orexperimentally, as outlined herein. A PDA™ variant TNF-alpha library maybe combined with a sequence alignment variant TNF-alpha library, andeither recombined (again, computationally or experimentally) or just thecutoffs from each joined to make a new tertiary library. The topsequences from several libraries may be recombined. Sequences from thetop of a library may be combined with sequences from the bottom of thelibrary to more broadly sample sequence space, or only sequences distantfrom the top of the library may be combined. Variant TNF-alpha librariesthat analyzed different parts of a protein may be combined to a tertiarylibrary that treats the combined parts of the protein.

In a preferred embodiment, a tertiary library may be generated usingcorrelations in a variant TNF-alpha library. That is, a residue at afirst variable position may be correlated to a residue at secondvariable position (or correlated to residues at additional positions aswell). For example, two variable positions may sterically orelectrostatically interact, such that if the first residue is X, thesecond residue must be Y. This may be either a positive or negativecorrelation.

Using the nucleic acids of the present invention which encode a variantTNF-alpha protein, a variety of expression vectors are made. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the variantTNF-alpha protein. The term “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation.

In a preferred embodiment, when the endogenous secretory sequence leadsto a low level of secretion of the naturally occurring protein or of thevariant TNF-alpha protein, a replacement of the naturally occurringsecretory leader sequence is desired. In this embodiment, an unrelatedsecretory leader sequence is operably linked to a variant TNF-alphaencoding nucleic acid leading to increased protein secretion. Thus, anysecretory leader sequence resulting in enhanced secretion of the variantTNF-alpha protein, when compared to the secretion of TNF-alpha and itssecretory sequence, is desired. Suitable secretory leader sequences thatlead to the secretion of a protein are known in the art.

In another preferred embodiment, a secretory leader sequence of anaturally occurring protein or a protein is removed by techniques knownin the art and subsequent expression results in intracellularaccumulation of the recombinant protein.

Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading frame. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. Thetranscriptional and translational regulatory nucleic acid will generallybe appropriate to the host cell used to express the fusion protein; forexample, transcriptional and translational regulatory nucleic acidsequences from Bacillus are preferably used to express the fusionprotein in Bacillus. Numerous types of appropriate expression vectors,and suitable regulatory sequences are known in the art for a variety ofhost cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention. In a preferred embodiment, the promoters are strongpromoters, allowing high expression in cells, particularly mammaliancells, such as the CMV promoter, particularly in combination with a Tetregulatory element.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference. In a preferredembodiment, the expression vector comprises the components describedabove and a gene encoding a variant TNF-alpha protein. As will beappreciated by those in the art, all combinations are possible andaccordingly, as used herein, the combination of components, comprised byone or more vectors, which may be retroviral or not, is referred toherein as a “vector composition”.

The variant TNF-alpha nucleic acids are introduced into the cells eitheralone or in combination with an expression vector. By “introduced into”or grammatical equivalents herein is meant that the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type, discussed below. Exemplary methods include CaPO₄precipitation, liposome fusion, lipofectin®, electroporation, viralinfection, etc. The variant TNF-alpha nucleic acids may stably integrateinto the genome of the host cell (for example, with retroviralintroduction, outlined below), or may exist either transiently or stablyin the cytoplasm (i.e. through the use of traditional plasmids,utilizing standard regulatory sequences, selection markers, etc.).

The variant TNF-alpha proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a variant TNF-alpha protein, under the appropriateconditions to induce or cause expression of the variant TNF-alphaprotein. The conditions appropriate for variant TNF-alpha proteinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the artthrough routine experimentation.

For example, the use of constitutive promoters in the expression vectorwill require optimizing the growth and proliferation of the host cell,while the use of an inducible promoter requires the appropriate growthconditions for induction. In addition, in some embodiments, the timingof the harvest is important. For example, the baculoviral systems usedin insect cell expression are lytic viruses, and thus harvest timeselection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melangaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc.

In a preferred embodiment, the variant TNF-alpha proteins are expressedin mammalian cells. Mammalian expression systems are also known in theart, and include retroviral systems. A mammalian promoter is any DNAsequence capable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for the fusionprotein into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and a TATA box, using a located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A mammalianpromoter will also contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as mammalian promoters are the promoters from mammalianviral genes, since the viral genes are often highly expressed and have abroad host range. Examples include the SV40 early promoter, mousemammary tumor virus LTR promoter, adenovirus major late promoter, herpessimplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei. As outlined herein, a particularly preferred methodutilizes retroviral infection, as outlined in PCT US97/01019,incorporated by reference.

As will be appreciated by those in the art, the type of mammalian cellsused in the present invention can vary widely. Basically, any mammaliancells may be used, with mouse, rat, primate and human cells beingparticularly preferred, although as will be appreciated by those in theart, modifications of the system by pseudotyping allows all eukaryoticcells to be used, preferably higher eukaryotes. As is more fullydescribed below, a screen will be set up such that the cells exhibit aselectable phenotype in the presence of a bioactive peptide. As is morefully described below, cell types implicated in a wide variety ofdisease conditions are particularly useful, so long as a suitable screenmay be designed to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a peptide within the cell.

Accordingly, suitable cell types include, but are not limited to, tumorcells of all types (particularly melanoma, myeloid leukemia, carcinomasof the lung, breast, ovaries, colon, kidney, prostate, pancreas andtestes), cardiomyocytes, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell), mast cells, eosinophils, vascularintimal cells, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoietic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

In one embodiment, the cells may be additionally genetically engineered,that is, contain exogenous nucleic acid other than the variant TNF-alphanucleic acid.

In a preferred embodiment, the variant TNF-alpha proteins are expressedin bacterial systems. Bacterial expression systems are well known in theart.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of the variant TNF-alpha proteininto mRNA. A bacterial promoter has a transcription initiation regionwhich is usually placed proximal to the 5′ end of the coding sequence.This transcription initiation region typically includes an RNApolymerase binding site and a transcription initiation site. Sequencesencoding metabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter may include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon. The expression vector may also includea signal peptide sequence that provides for secretion of the variantTNF-alpha protein in bacteria. The signal sequence typically encodes asignal peptide comprised of hydrophobic amino acids which direct thesecretion of the protein from the cell, as is well known in the art. Theprotein is either secreted into the growth media (gram-positivebacteria) or into the periplasmic space, located between the inner andouter membrane of the cell (gram-negative bacteria). For expression inbacteria, usually bacterial secretory leader sequences, operably linkedto a variant TNF-alpha encoding nucleic acid, are preferred.

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, variant TNF-alpha proteins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, variant TNF-alpha protein is produced inyeast cells. Yeast expression systems are well known in the art, andinclude expression vectors for Saccharomyces cerevisiae, Candidaalbicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilisand K. lactis, Pichia guillerimondii and P. pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred promotersequences for expression in yeast include the inducible GAL1,10promoter, the promoters from alcohol dehydrogenase, enolase,glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

In a preferred embodiment, modified TNF variants are covalently coupledto at least one additional TNF variant via a linker to improve thedominant negative action of the modified domains. A number of strategiesmay be used to covalently link modified receptor domains together. Theseinclude, but are not limited to, linkers, such as polypeptide linkagesbetween N- and C-termini of two domains, linkage via a disulfide bondbetween monomers, and linkage via chemical cross-linking reagents.Alternatively, the N- and C-termini may be covalently joined by deletionof portions of the N- and/or C-termini and linking the remainingfragments via a linker or linking the fragments directly.

By “linker”, “linker sequence”, “spacer”, “tethering sequence” orgrammatical equivalents thereof, herein is meant a molecule or group ofmolecules (such as a monomer or polymer) that connects two molecules andoften serves to place the two molecules in a preferred configuration. Inone aspect of this embodiment, the linker is a peptide bond. Choosing asuitable linker for a specific case where two polypeptide chains are tobe connected depends on various parameters, e.g., the nature of the twopolypeptide chains (e.g., whether they naturally oligomerize (e.g., forma dimer or not), the distance between the N- and the C-termini to beconnected if known from three-dimensional structure determination,and/or the stability of the linker towards proteolysis and oxidation.Furthermore, the linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. These linkedTNF-alpha proteins have constrained hydrodynamic properties, that is,they form constitutive dimers) and thus efficiently interact with othernaturally occurring TNF-alpha proteins to form a dominant negativeheterotrimer.

The linker peptide should have a length that is adequate to link two TNFvariant monomers in such a way that they assume the correct conformationrelative to one another so that they retain the desired activity asantagonists of the TNF receptor. Suitable lengths for this purposeinclude at least one and not more than 30 amino acid residues.Preferably, the linker is from about 1 to 30 amino acids in length, withlinkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1819 and 20 amino acids in length being preferred. See also WO 01/25277,incorporated herein by reference in its entirety.

In addition, the amino acid residues selected for inclusion in thelinker peptide should exhibit properties that do not interferesignificantly with the activity of the polypeptide. Thus, the linkerpeptide on the whole should not exhibit a charge that would beinconsistent with the activity of the polypeptide, or interfere withinternal folding, or form bonds or other interactions with amino acidresidues in one or more of the monomers that would seriously impede thebinding of receptor monomer domains.

Useful linkers include glycine-serine polymers (including, for example,(GS)_(n), (GSGGS)_(n) (SEQ ID NO:31) (GGGGS)_(n) (SEQ ID NO:32) and(GGGS)_(n), (SEQ ID NO:33), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers such as the tether for the shaker potassium channel, and a largevariety of other flexible linkers, as will be appreciated by those inthe art. Glycine-serine polymers are preferred since both of these aminoacids are relatively unstructured, and therefore may be able to serve asa neutral tether between components. Secondly, serine is hydrophilic andtherefore able to solubilize what could be a globular glycine chain.Third, similar chains have been shown to be effective in joiningsubunits of recombinant proteins such as single chain antibodies.

Suitable linkers may also be identified by screening databases of knownthree-dimensional structures for naturally occurring motifs that canbridge the gap between two polypeptide chains. Another way of obtaininga suitable linker is by optimizing a simple linker, e.g., (Gly4Ser)_(n),(SEQ ID NO:32), through random mutagenesis. Alternatively, once asuitable polypeptide linker is defined, additional linker polypeptidescan be created by application of PDA™ technology to select amino acidsthat more optimally interact with the domains being linked. Other typesof linkers that may be used in the present invention include artificialpolypeptide linkers and inteins. In another preferred embodiment,disulfide bonds are designed to link the two receptor monomers atinter-monomer contact sites. In one aspect of this embodiment the tworeceptors are linked at distances<5 Angstroms. In addition, the variantTNF-alpha polypeptides of the invention may be further fused to otherproteins, if desired, for example to increase expression or stabilizethe protein.

In one embodiment, the variant TNF-alpha nucleic acids, proteins andantibodies of the invention are labeled with a label other than thescaffold. By “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) immune labels,which may be antibodies or antigens; and c) colored or fluorescent dyes.The labels may be incorporated into the compound at any position.

Once made, the variant TNF-alpha proteins may be covalently modified.Covalent and non-covalent modifications of the protein are thus includedwithin the scope of the present invention. Such modifications may beintroduced into a variant TNF-alpha polypeptide by reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues.

One type of covalent modification includes reacting targeted amino acidresidues of a variant TNF-alpha polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of a variant TNF-alpha polypeptide. Derivatizationwith bifunctional agents is useful, for instance, for cross linking avariant TNF-alpha protein to a water-insoluble support matrix or surfacefor use in the method for purifying anti-variant TNF-alpha antibodies orscreening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio] propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the variant TNF-alphapolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencevariant TNF-alpha polypeptide, and/or adding one or more glycosylationsites that are not present in the native sequence variant TNF-alphapolypeptide.

Addition of glycosylation sites to variant TNF-alpha polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence or variantTNF-alpha polypeptide (for O-linked glycosylation sites). The variantTNF-alpha amino acid sequence may optionally be altered through changesat the DNA level, particularly by mutating the DNA encoding the variantTNF-alpha polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Addition of N-linked glycosylation sites to variant TNF-alphapolypeptides may be accomplished by altering the amino acid sequencethereof. The alteration may be made, for example, by the addition of, orsubstitution by, one or more asparagine residues to the native sequenceor variant TNF-alpha polypeptide. The modification may be made forexample by the incorporation of a canonical N-linked glycosylation site,including but not limited to, N-X-Y, where X is any amino acid exceptfor proline and Y is preferably threonine, serine or cysteine. Anothermeans of increasing the number of carbohydrate moieties on the variantTNF-alpha polypeptide is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, C R C Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the variant TNF-alphapolypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo-andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Such derivatized moieties may improve the solubility, absorption, andpermeability across the blood brain barrier biological half life, andthe like. Such moieties or modifications of variant TNF-alphapolypeptides may alternatively eliminate or attenuate any possibleundesirable side effect of the protein and the like. Moieties capable ofmediating such effects are disclosed, for example, in Remington'sPharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.(1980).

Another type of covalent modification of variant TNF-alpha compriseslinking the variant TNF-alpha polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. These nonproteinaceous polymers may also be used to enhancethe variant TNF-alpha's ability to disrupt receptor binding, and/or invivo stability.

In another preferred embodiment, cysteines are designed into variant orwild-type TNF-alpha in order to incorporate (a) labeling sites forcharacterization and (b) incorporate PEGylation sites. For example,labels that may be used are well known in the art and include but arenot limited to biotin, tag and fluorescent labels (e.g. fluorescein).These labels may be used in various assays as are also well known in theart to achieve characterization. A variety of coupling chemistries maybe used to achieve PEGylation, as is well known in the art. Examples,include but are not limited to, the technologies of Shearwater andEnzon, which allow modification at primary amines, including but notlimited to, lysine groups and the N-terminus. See, Kinstler et al,Advanced Drug Deliveries Reviews, 54, 477-485 (2002) and M J Roberts etal, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both herebyincorporated by reference.

Optimal sites for modification can be chosen using a variety ofcriteria, including but not limited to, visual inspection, structuralanalysis, sequence analysis and molecular simulation. For example, asshown in FIG. 18, the fractional accessibility (surface_aa) ofindividual residues was analyzed to identify mutational sites that willnot disrupt the monomer structure. Then the minimum distance(mindistance) from each side chain of a monomer to another subunit wascalculated to ensure that chemical modification will not disrupttrimerization. It is possible that receptor binding disruption may occurand may be beneficial to the activity of the TNF variants of thisinvention.

In a preferred embodiment, the optimal chemical modification sites forthe TNF-alpha variants of the present invention, include but are notlimited to:

<surface> <mindistance> <combined> GLU 23 0.9 0.9 0.8 GLN 21 0.8 0.9 0.7ASP 45 0.7 1.0 0.7 ASP 31 0.8 0.6 0.5 ARG 44 0.6 0.9 0.5 GLN 25 0.5 1.00.5 GLN 88 0.7 0.7 0.4 GLY 24 0.5 0.9 0.4 ASP 140 0.6 0.7 0.4 GLU 42 0.50.8 0.4 GLU 110 0.8 0.4 0.4 GLY 108 0.8 0.4 0.3 GLN 27 0.4 0.9 0.3 GLU107 0.7 0.4 0.3 ASP 10 0.7 0.4 0.3 SER 86 0.6 0.5 0.3 ALA 145 0.8 0.40.3 LYS 128 0.6 0.4 0.3 ASN 46 0.3 0.9 0.3 LYS 90 0.5 0.5 0.3 TYR 87 0.60.4 0.3

In a more preferred embodiment, the optimal chemical modification sitesare 21, 23, 31 and 45, taken alone or in any combination.

In a another preferred embodiment, portions of either the N- orC-termini of the wild type TNF-alpha monomer are deleted while stillallowing the TNF-alpha molecule to fold properly. In addition, thesemodified TNF-alpha proteins would lack receptor binding ability, andcould optionally interact with other wild type TNF alpha molecules ormodified TNF-alpha proteins to form trimers as described above. Morespecifically, removal or deletion of from about 1 to about 55 aminoacids from either the N or C termini, or both, are preferred. A morepreferred embodiment includes deletions of N-termini beyond residue 10and more preferably, deletion of the first 47 N-terminal amino acids.The deletion of C-terminal leucine is an alternative embodiment.

In another preferred embodiment, the wild type TNF-alpha or variantsgenerated by the invention may be circularly permuted. All naturalproteins have an amino acid sequence beginning with an N-terminus andending with a C-terminus. The N- and C-termini may be joined to create acyclized or circularly permutated TNF-alpha proteins while retaining orimproving biological properties (e.g., such as enhanced stability andactivity) as compared to the wild-type protein. In the case of aTNF-alpha protein, a novel set of N- and C-termini are created at aminoacid positions normally internal to the protein's primary structure, andthe original N- and C-termini are joined via a peptide linker consistingof from 0 to 30 amino acids in length (in some cases, some of the aminoacids located near the original termini are removed to accommodate thelinker design). In a preferred embodiment, the novel N- and C-terminiare located in a non-regular secondary structural element, such as aloop or turn, such that the stability and activity of the novel proteinare similar to those of the original protein. The circularly permutedTNF-alpha protein may be further PEGylated or glycosylated. In a furtherpreferred embodiment PDA™ technology may be used to further optimize theTNF-alpha variant, particularly in the regions created by circularpermutation. These include the novel N- and C-termini, as well as theoriginal termini and linker peptide.

Various techniques may be used to permutate proteins. See U.S. Pat. No.5,981,200; Maki K, Iwakura M., Seikagaku. 2001 January; 73(1): 42-6; PanT., Methods Enzymol. 2000; 317:313-30; Heinemann U, Hahn M., ProgBiophys Mol. Biol. 1995; 64(2-3): 121-43; Harris M E, Pace N R, Mol BiolRep. 1995-96; 22(2-3):115-23; Pan T, Uhlenbeck O C., Mar. 30, 1993;125(2): 111-4; Nardulli A M, Shapiro D J. 1993 Winter; 3(4):247-55, EP1098257 A2; WO 02/22149; WO 01/51629; WO 99/51632; Hennecke, et al.,1999, J. Mol. Biol., 286, 1197-1215; Goldenberg et al J. Mol. Biol 165,407-413 (1983); Luger et al, Science, 243, 206-210 (1989); and Zhang etal., Protein Sci 5, 1290-1300 (1996); all hereby incorporated byreference.

In addition, a completely cyclic TNF-alpha may be generated, wherein theprotein contains no termini. This is accomplished utilizing inteintechnology. Thus, peptides can be cyclized and in particular inteins maybe utilized to accomplish the cyclization.

Variant TNF-alpha polypeptides of the present invention may also bemodified in a way to form chimeric molecules comprising a variantTNF-alpha polypeptide fused to another, heterologous polypeptide oramino acid sequence. In one embodiment, such a chimeric moleculecomprises a fusion of a variant TNF-alpha polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the variant TNF-alpha polypeptide. The presence ofsuch epitope-tagged forms of a variant TNF-alpha polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the variant TNF-alpha polypeptide to bereadily purified by affinity purification using an anti-tag antibody oranother type of affinity matrix that binds to the epitope tag. In analternative embodiment, the chimeric molecule may comprise a fusion of avariant TNF-alpha polypeptide with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule, such a fusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A.87:6393-6397 (1990)].

In a preferred embodiment, the variant TNF-alpha protein is purified orisolated after expression. Variant TNF-alpha proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the variant TNF-alpha protein may be purified using astandard anti-library antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the variantTNF-alpha protein. In some instances no purification will be necessary.

Once made, the variant TNF-alpha proteins and nucleic acids of theinvention find use in a number of applications. In a preferredembodiment, the variant TNF-alpha proteins are administered to a patientto treat an TNF-alpha related disorder.

By “TNF-α related disorder” or “TNF-alpha responsive disorder” or“condition” herein is meant a disorder that may be ameliorated by theadministration of a pharmaceutical composition comprising a variantTNF-alpha protein, including, but not limited to, inflammatory andimmunological disorders. The variant TNF-alpha is a major effector andregulatory cytokine with a pleiotropic role in the pathogenesis ofimmune-regulated diseases.

In a preferred embodiment, the variant TNF-alpha protein is used totreat spondyloarthritis, rheumatoid arthritis, inflammatory boweldiseases, sepsis and septic shock, Crohn's Disease, psoriasis, graftversus host disease (GVHD) and hematologic malignancies, such asmultiple myeloma (MM), myelodysplastic syndrome (MDS) and acutemyelogenous leukemia (AML), cancer and the inflammation associated withtumors, peripheral nerve injury or demyelinating diseases. See, forexample, Tsimberidou et al., Expert Rev Anticancer Ther 2002 June;2(3):277-86.

Inflammatory bowel disease (“IBD”) is the term generally applied to twodiseases, namely ulcerative colitis and Crohn's disease. Ulcerativecolitis is a chronic inflammatory disease of unknown etiology afflictingonly the large bowel and, except when very severe, limited to the bowelmucosa. The course of the disease may be continuous or relapsing, mildor severe. It is curable by total colostomy which may be needed foracute severe disease or chronic unremitting disease.

Crohn's disease is also a chronic inflammatory disease of unknownetiology but, unlike ulcerative colitis, it can affect any part of thebowel. Although lesions may start superficially, the inflammatoryprocess extends through the bowel wall to the draining lymph nodes. Aswith ulcerative colitis, the course of the disease may be continuous orrelapsing, mild or severe but, unlike ulcerative colitis, it is notcurable by resection of the involved segment of bowel. Most patientswith Crohn's disease come to surgery at some time, but subsequentrelapse is common and continuous medical treatment is usual.

Remicade® (inflixmab) is the commercially available treatment forCrohn's disease. Remicade® is a chimeric monoclonal antibody that bindsto TNF-alpha. The use of the TNF-alpha variants of the present inventionmay also be used to treat the conditions associated with IBD or Crohn'sDisease.

“Sepsis” is herein defined to mean a disease resulting from grampositive or gram negative bacterial infection, the latter primarily dueto the bacterial endotoxin, lipopolysaccharide (LPS). It can be inducedby at least the six major gram-negative bacilli and these arePseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella,Enterobacter and Serratia.

Septic shock is a condition which may be associated with Gram positiveinfections, such as those due to pneumococci and streptococci, or withGram negative infections, such as those due to Escherichia coli,Klebsiella-Enterobacter, Pseudomonas, and Serratia. In the case of theGram-negative organisms the shock syndrome is not due to bloodstreaminvasion with bacteria per se but is related to release of endotoxin,the LPS moiety of the organisms' cell walls, into the circulation.Septic shock is characterized by inadequate tissue perfusion andcirculatory insufficiency, leading to insufficient oxygen supply totissues, hypotension, tachycardia, tachypnea, fever and oliguria. Septicshock occurs because bacterial products, principally LPS, react withcell membranes and components of the coagulation, complement,fibrinolytic, bradykinin and immune systems to activate coagulation,injure cells and alter blood flow, especially in the microvasculature.Microorganisms frequently activate the classic complement pathway, andendotoxin activates the alternate pathway.

The TNF-alpha variants of the present invention effectively antagonizethe effects of wild type TNF-alpha-induced cytotoxicity and interferewith the conversion of TNF into a mature TNF molecule (e.g. the trimerform of TNF). Thus, administration of the TNF variants can ameliorate oreliminate the effects of sepsis or septic shock, as well as inhibit thepathways associated with sepsis or septic shock. Administration may betherapeutic or prophylactic.

The TNF-alpha variants of the present invention effectively antagonizethe effects of wild type TNF-alpha-induced cytotoxicity in cell basedassays and animal models of peripheral nerve injury and axonaldemyelination/degeneration to reduce the inflammatory component of theinjury or demyelinating insult. This is believed to criticallycontribute to the neuropathological and behavioral sequelae andinfluence the pathogenesis of painful neuropathies.

Severe nerve injury induces activation of Matrix Metallo Proteinases(MMPs), including TACE, the TNF-alpha-converting enzyme, resulting inelevated levels of TNF-alpha protein at an early time point in thecascade of events that leads up to Wallerian nerve degeneration andincreased pain sensation (hyperalgesia). The TNF-alpha variants of thepresent invention antagonize the activity of these elevated levels ofTNF-alpha at the site of peripheral nerve injury with the intent ofreducing macrophage recruitment from the periphery without negativelyaffecting remyelination. Thus, reduction of local TNF-inducedinflammation with these TNF-alpha variants would represent a therapeuticstrategy in the treatment of the inflammatory demyelination and axonaldegeneration in peripheral nerve injury as well as the chronichyperalgesia characteristic of neuropathic pain states that oftenresults from such peripheral nerve injuries.

Intraneural administration of exogenous TNF-alpha produces inflammatoryvascular changes within the lining of peripheral nerves (endoneurium)together with demyelination and axonal degeneration (Redford et al1995). After nerve transection, TNF-positive macrophages can be foundwithin degenerating fibers and are believed to be involved in myelindegradation after axotomy (Stoll et al 1993). Furthermore, peripheralnerve glia (Schwann cells) and endothelial cells produce extraordinaryamounts of TNF-alpha at the site of nerve injury (Wagner et al 1996) andintraperitoneal application of anti-TNF antibody significantly reducesthe degree of inflammatory demyelination strongly implicating apathogenic role for TNF-alpha in nerve demyelination and degeneration(Stoll et al., 1993). Thus, administration of an effective amount of theTNF-alpha variants of the present invention may be used to treat theseperipheral nerve injury or demyelinating conditions. In a preferredembodiment, a therapeutically effective dose of a variant TNF-alphaprotein is administered to a patient in need of treatment. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. In a preferred embodiment, dosages ofabout 5 μg/kg are used, administered either intravenously orsubcutaneously. As is known in the art, adjustments for variantTNF-alpha protein degradation, systemic versus localized delivery, andrate of new protease synthesis, as well as the age, body weight, generalhealth, sex, diet, time of administration, drug interaction and theseverity of the condition may be necessary, and will be ascertainablewith routine experimentation by those skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The term “treatment” in the instant invention is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor the disease or disorder. Thus, for example, successfuladministration of a variant TNF-alpha protein prior to onset of thedisease results in “treatment” of the disease. As another example,successful administration of a variant TNF-alpha protein after clinicalmanifestation of the disease to combat the symptoms of the diseasecomprises “treatment” of the disease. “Treatment” also encompassesadministration of a variant TNF-alpha protein after the appearance ofthe disease in order to eradicate the disease. Successful administrationof an agent after onset and after clinical symptoms have developed, withpossible abatement of clinical symptoms and perhaps amelioration of thedisease, comprises “treatment” of the disease.

Those “in need of treatment” include mammals already having the diseaseor disorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In another embodiment, a therapeutically effective dose of a variantTNF-alpha protein, a variant TNF-alpha gene, or a variant TNF-alphaantibody is administered to a patient having a disease involvinginappropriate expression of TNF-alpha. A “disease involvinginappropriate expression of at TNF-alpha” within the scope of thepresent invention is meant to include diseases or disorderscharacterized by aberrant TNF-alpha, either by alterations in the amountof TNF-alpha present or due to the presence of mutant TNF-alpha. Anoverabundance may be due to any cause, including, but not limited to,overexpression at the molecular level, prolonged or accumulatedappearance at the site of action, or increased activity of TNF-alpharelative to normal. Included within this definition are diseases ordisorders characterized by a reduction of TNF-alpha. This reduction maybe due to any cause, including, but not limited to, reduced expressionat the molecular level, shortened or reduced appearance at the site ofaction, mutant forms of TNF-alpha, or decreased activity of TNF-alpharelative to normal. Such an overabundance or reduction of TNF-alpha canbe measured relative to normal expression, appearance, or activity ofTNF-alpha according to, but not limited to, the assays described andreferenced herein.

The administration of the variant TNF-alpha proteins of the presentinvention, preferably in the form of a sterile aqueous solution, may bedone in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds, inflammation, etc., the variant TNF-alpha protein may bedirectly applied as a solution or spray. Depending upon the manner ofintroduction, the pharmaceutical composition may be formulated in avariety of ways. The concentration of the therapeutically active variantTNF-alpha protein in the formulation may vary from about 0.1 to 100weight %. In another preferred embodiment, the concentration of thevariant TNF-alpha protein is in the range of 0.003 to 1.0 molar, withdosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram ofbody weight being preferred.

The pharmaceutical compositions of the present invention comprise avariant TNF-alpha protein in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers such asNaOAc; fillers such as microcrystalline cellulose, lactose, corn andother starches; binding agents; sweeteners and other flavoring agents;coloring agents; and polyethylene glycol. Additives are well known inthe art, and are used in a variety of formulations.

In a further embodiment, the variant TNF-alpha proteins are added in amicellular formulation; see U.S. Pat. No. 5,833,948, hereby expresslyincorporated by reference in its entirety.

Combinations of pharmaceutical compositions may be administered.Moreover, the compositions may be administered in combination with othertherapeutics.

In one embodiment provided herein, antibodies, including but not limitedto monoclonal and polyclonal antibodies, are raised against variantTNF-alpha proteins using methods known in the art. In a preferredembodiment, these anti-variant TNF-alpha antibodies are used forimmunotherapy. Thus, methods of immunotherapy are provided. By“immunotherapy” is meant treatment of an TNF-alpha related disorderswith an antibody raised against a variant TNF-alpha protein. As usedherein, immunotherapy can be passive or active. Passive immunotherapy,as defined herein, is the passive transfer of antibody to a recipient(patient). Active immunization is the induction of antibody and/orT-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with avariant TNF-alpha protein antigen to which antibodies are raised. Asappreciated by one of ordinary skill in the art, the variant TNF-alphaprotein antigen may be provided by injecting a variant TNF-alphapolypeptide against which antibodies are desired to be raised into arecipient, or contacting the recipient with a variant TNF-alpha proteinencoding nucleic acid, capable of expressing the variant TNF-alphaprotein antigen, under conditions for expression of the variantTNF-alpha protein antigen.

In another preferred embodiment, a therapeutic compound is conjugated toan antibody, preferably an anti-variant TNF-alpha protein antibody. Thetherapeutic compound may be a cytotoxic agent. In this method, targetingthe cytotoxic agent to tumor tissue or cells, results in a reduction inthe number of afflicted cells, thereby reducing symptoms associated withcancer, and variant TNF-alpha protein related disorders. Cytotoxicagents are numerous and varied and include, but are not limited to,cytotoxic drugs or toxins or active fragments of such toxins. Suitabletoxins and their corresponding fragments include diphtheria A chain,exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin,phenomycin, enomycin and the like. Cytotoxic agents also includeradiochemicals made by conjugating radioisotopes to antibodies raisedagainst cell cycle proteins, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody.

In a preferred embodiment, variant TNF-alpha proteins are administeredas therapeutic agents, and can be formulated as outlined above.Similarly, variant TNF-alpha genes (including both the full-lengthsequence, partial sequences, or regulatory sequences of the variantTNF-alpha coding regions) may be administered in gene therapyapplications, as is known in the art. These variant TNF-alpha genes caninclude antisense applications, either as gene therapy (i.e. forincorporation into the genome) or as antisense compositions, as will beappreciated by those in the art.

In a preferred embodiment, the nucleic acid encoding the variantTNF-alpha proteins may also be used in gene therapy. In gene therapyapplications, genes are introduced into cells in order to achieve invivo synthesis of a therapeutically effective genetic product, forexample for replacement of a defective gene. “Gene therapy” includesboth conventional gene therapy where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. Antisense RNAs and DNAs can beused as therapeutic agents for blocking the expression of certain genesin vivo. It has already been shown that short antisense oligonucleotidescan be imported into cells where they act as inhibitors, despite theirlow intracellular concentrations caused by their restricted uptake bythe cell membrane. [Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A.83:4143-4146 (1986)]. The oligonucleotides can be modified to enhancetheir uptake, e.g. by substituting their negatively chargedphosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. U.S.A. 87:3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science 256:808-813 (1992).

In a preferred embodiment, variant TNF-alpha genes are administered asDNA vaccines, either single genes or combinations of variant TNF-alphagenes. Naked DNA vaccines are generally known in the art. Brower, NatureBiotechnology, 16:1304-1305 (1998). Methods for the use of genes as DNAvaccines are well known to one of ordinary skill in the art, and includeplacing a variant TNF-alpha gene or portion of a variant TNF-alpha geneunder the control of a promoter for expression in a patient in need oftreatment.

The variant TNF-alpha gene used for DNA vaccines can encode full-lengthvariant TNF-alpha proteins, but more preferably encodes portions of thevariant TNF-alpha proteins including peptides derived from the variantTNF-alpha protein. In a preferred embodiment a patient is immunized witha DNA vaccine comprising a plurality of nucleotide sequences derivedfrom a variant TNF-alpha gene. Similarly, it is possible to immunize apatient with a plurality of variant TNF-alpha genes or portions thereofas defined herein. Without being bound by theory, expression of thepolypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced which recognize and destroy oreliminate cells expressing TNF-alpha proteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the variantTNF-alpha polypeptide encoded by the DNA vaccine. Additional oralternative adjuvants are known to those of ordinary skill in the artand find use in the invention.

All references cited herein, including patents, patent applications(provisional, utility and PCT), and publications are incorporated byreference in their entirety.

EXAMPLES Example 1 Protocol for TNF-alpha Library Expression,Purification, and Activity Assays for TNF-alpha Variants

Methods:

1) Overnight Culture Preparation:

Competent Tuner(DE3)pLysS cells in 96 well-PCR plates were transformedwith 1 ul of TNF-alpha library DNAs and spread on LB agar plates with 34mg/ml chloramphenicol and 100 mg/ml ampicillin. After an overnightgrowth at 37 degrees C., a colony was picked from each plate in 1.5 mlof CG media with 34 mg/ml chloramphenicol and 100 mg/ml ampicillin keptin 96 deep well block. The block was shaken at 250 rpm at 37 degrees C.overnight.

2) Expression:

Colonies were picked from the plate into 5 ml CG media (34 mg/mlchloramphenicol and 100 mg/ml ampicillin) in 24-well block and grown at37 degrees C. at 250 rpm until OD600 0.6 were reached, at which timeIPTG was added to each well to 1 mM concentration. The culture was grown4 extra hours.

3) Lysis:

The 24-well block was centrifuged at 3000 rpm for 10 minutes. Thepellets were resuspended in 700 ul of lysis buffer (50 mM NaH2PO4, 300mM NaCl, 10 mM imidazole). After freezing at −80 degrees C. for 20minutes and thawing at 37 degrees C. twice, MgCl2 was added to 10 mM,and DNase I to 75 mg/ml. The mixture was incubated at 37 degrees C. for30 minutes.

4) Ni NTA Column Purification:

Purification was carried out following Qiagen Ni NTA spin columnpurification protocol for native condition. The purified protein wasdialyzed against 1×PBS for 1 hour at 4 degrees C. four times. Dialyzedprotein was filter sterilized, using Millipore multiscreenGV filterplate to allow the addition of protein to the sterile mammalian cellculture assay later on.

5) Quantification:

Purified protein was quantified by SDS PAGE, followed by Coomassiestain, and by Kodak® digital image densitometry.

6) TNF-Alpha Activity Assay Assays:

The activity of variant TNF-alpha protein samples was tested usingVybrant Assay Kit and Caspase Assay kit. Sytox Green nucleic acid stainis used to detect TNF-induced cell permeability in Actinomycin-Dsensitized cell line. Upon binding to cellular nucleic acids, the stainexhibits a large fluorescence enhancement, which is then measured. Thisstain is excluded from live cells but penetrates cells with compromisedmembranes.

The caspase assay is a fluorimetric assay, which can differentiatebetween apoptosis and necrosis in the cells. Cell extracts were madefrom cells treated to induce apoptosis. These extracts were supplementedwith a fluorescently-conjugated caspase substrate (DEVD-R110) peptide.Activated caspase enzymes cleave the DEVD-R110 peptide to produce afluorescent enhancement of R110. Therefore, R110 fluorescence is adirect measure of caspase activity, which is a direct measure ofapoptosis.

A) Materials:

Cell Line: WEHI Var-13 Cell line from ATCC Media: RPMI Complete mediawith 10% FBS. Vybrant TNF Kit: Cat # V-23100; Molecular Probes

-   Kit contains SYTOX Green nucleic acid stain (500 mM solution)-   and Actinomycin D (1 mg/mL)-   Caspase Assay Kit: Cat #3 005 372; Roche    -   Kit contains substrate stock solution (500 uM)-   and incubation buffer-   TNF-alpha Standard stock: 10 ug/mL stock of h-TNF-alpha from R & D-   Unknown Samples: In house TNF-alpha library samples-   96-well Plates: 1 mL deep well and 250 m wells-   Micro plate Reader    B) Method:

Plate WEHI164-13Var cells at 2.5×105 cells/mL in full RPMI medium, 24hrs prior to the assay; (100 uL/well for the Sytox assay and 50 uL/wellfor the Caspase assay).

On the day of the experiment, prepare assay media as follows:

-   1) Assay Media for Sytox Assay (1×): Prepare assay medium by    diluting the concentrated Sytox Green stain and the concentrated    actinomycin D solution 500-fold into RPMI, to a final concentration    of 10 mM Sytox and 2 mg/mL actinomycin D.    -   10 mL complete RPMI medium    -   20 mL SYTOX Green    -   20 mL actinomycin D-   2) Prepare Assay Media for Caspase Assay (1×):    -   10 mL complete RPMI medium    -   20 uL Actinomycin D (2 mg/mL final conc.)-   3) Prepare Assay Media for samples: Sytox Assay (2×):    -   14 mL complete RPMI medium    -   56 mL SYTOX Green Nuclei acid stain    -   56 mL actinomycin D-   4) Prepare Assay Media: (2×): For samples: Caspase assay    -   14 mL complete RPMI medium    -   56 mL actinomycin D-   5) Set up and Run a Standard Curve Dilution:    -   TNF-alpha Std. stock: 10 mg/mL    -   Dulute to 1 ug/mL: 10 mL stock+90 mL Assay medium.

1× Assay medium Conc. in Final Conc. for Sytox and dilution of TNF alphaStock (uL) Caspase (mL) plate on cells  10 uL of 1 mg 990 10 ng/mL 5ng/mL  5 uL of 1 mg 995 5 ng/mL 2.5 ng/mL 200 uL of 5 ng 300 2 ng/mL 1ng/mL 100 uL of 5 ng 400 1 ng/mL 0.5 ng/mL 100 uL of 5 ng 900 500 pg/mL250 pg/mL 200 uL of 500 pg 300 200 pg/mL 100 pg/mL 100 uL of 500 pg 400100 pg/mL 50 pg/mL  50 uL of 500 pg 450 50 pg/mL 25 pg/mL  20 uL of 500pg 480 20 pg/mL 10 pg/mL  10 uL of 500 pg 490 10 pg/mL 5 pg/mL  0 uL 5000 pg/mL 0 pg/mL

For Unknown Samples: (Quantitated by Gel): TNF-alpha Library:

Normalize all the samples to the same starting concentration (500 ng/mL)as follows:

-   -   Neat: 500 ng/mL: 100 mL    -   1:10 of 500 ng=50 ng/mL: 20 nL neat+180 mL RPMI    -   1:10 of 50 ng=5 ng/mL: 20 mL of 50 ng/mL+180 mL RPMI    -   1:10 of 5 ng/mL=0.5 ng/mL: 20 mL of 0.5 ng/mL+180 mL RPMI

-   6) For Sylox assay: On a separate dilution plate, add 60 mL of each    diluted sample to 60 mL of 2×Sylox assay media. Transfer 100 mL of    diluted samples to the cells cultured in 100 uL media. Incubate at    37 degrees C. for 6 hrs. Read the plate using a fluorescence    microplate reader with fillers appropriate for fluorescein (485 nm    excitation filter and 530 nm emission filter).

-   7) For Caspasc assay: On a separate dilution plate, add 35 mL of    each diluted sample to 35 mL of 2×Caspase assay media. Transfer 50    mL of dil. Samples to the cells cultured in 50 mL media. Incubate at    37 degrees C. for 4 hours. After 4 hrs. add Caspase Substrate (100    mL/well) [Predilute substrate 1:10]. Incubate 2 more hrs. at 37    degrees C. Read (fluorescence).    C) Data Analysis:

The fluorescence signal is directly proportional to the number ofapoptotic cells. Plot fluorescence vs. TNF-alpha standard concentrationto make a standard curve. Compare the fluorescence obtained from thehighest point on the standard curve (5 ng/mL) to the fluorescenceobtained from the unknown samples, to determine the percent activity ofthe samples.

The data may be analyzed using a four-parameter fit program to determinethe 50% effective concentration for TNF (EC50). Percent activity ofunknown samples=(Fluor. Of unknown samples/fluor. of 5 ng/mL std.Point)×100.

Example 2 TNF-alpha Activity Assay to Screen for Agonists of Wild TypeTNF-alpha Protein

A) Materials and Methods:

-   1) Plate cells for the TNF assay: WHEI plated at 2.5×105 Cells/ml    (50 μl /well in a 96 well plate).-   2) Prepare Assay Media as shown below:    -   a) 1×Assay Medium        -   10 ml complete RPMI        -   20 μl Actinomycin D    -   2×Assay Media:        -   7 ml complete RPMI medium        -   28 μl Actinomycin D-   3) Dilute TNF-Alpha Standards for Bioactiviy Assay: Requires two    standard Curves in duplicate as shown below:    -   In house TNF-alpha (lot #143-112stock: 1.1    -   Dilute to 40 μg/mL: 36 μl stock+964 μl assay medium.

Final Conc. Assay Conc. in TNF-alpha in Stock (μl) medium (μl) dilutionplate cells 500 ul of 40 ug/ml 500 20,000 ng/ml 10,000 ng/ml 500 ul of20,000 ng/ml 500 10,000 ng/ml 5,000 ng/ml 200 ul of 10,000 ng/ml 8002000 ng/ml 1000 ng/ml 500 ul of 2000 ng/ml 500 1000 ng/ml 500 ng/ml 200ul of 1000 ng/ml 800 200 ng/ml 100 ng/ml 500 ul of 200 ng/ml 500 100ng/ml 50 ng/ml 200 ul of 100 ng/ml 800 20 ng/ml 10 ng/ml  50 ul of 20ng/ml 950 1 ng/ml 0.5 ng/ml 200 ul of 1 ng/ml 800 0.2 ng/ml 0.1 ng/ml500 ul of 0.2 ng/ml 500 0.1 ng/ml 0.05 ng/ml 500 ul of 0.1 ng/ml 5000.05 ng/ml 0.025 ng/ml  0 500 0 0

-   4) Treatment of Unknown Samples from TNF-alpha Library:

Normalize all samples to the same starting concentration (200,000 ng/ml)by diluting samples as shown:

-   -   Neat: 200,000 ng/ml: 200 μl    -   1:10 of 200,000 ng/ml=20,000 ng/ml: 20 μl of neat+180 μl of RPMI    -   1:10 of 20,000 ng/ml=2000 ng/ml: 20 μl of 1:10+180 μl RPMI    -   1:10 of 2000 ng/ml=200 ng/ml: 20 μl of 1:100+180 μl RPMI    -   1:10 of 200 ng/ml=20 ng/ml: 20 μl of 1:100+180 μl RPMI

On a Separate Dilution Plate for Caspase Assay:

Add 150 μl of each diluted sample to 150 μl of 2×caspase assay media.

Incubate all the diluted samples and standard curve at 37° C. overnight.Next morning, transfer 50 μl of diluted samples to the cells with CM.After 4 hours prepare substrate, and then add 100 μl of substrate to thecells. Read fluorescence after 2 hours of incubation with substrate.

B) Results:

The results are summarized in FIG. 8.

Example 3 TNF-alpha Antagonist Activity

A) Materials and Methods:

-   1) Plate cells for the assay: WEHI plated at 2.5×105 cells/ml (50    μl/well)-   2) Prepare Assay Media:    -   1×Assay Medium”    -   40 ml complete RPMI medium    -   80 μl Actinomycin D (2 μg/ml final concentration)-   3) Antagonist Activity of TNF-alpha mutants”-   4) Preparation of assay medium+wild type TNF-alpha    -   Wild type TNF-alpha is 1.1 mg/ml    -   1 μg/ml: 1:1000; 1 μl of the stock in 1 ml of RPMI    -   20 ng/ml: 1:50 of the 1 μg/ml; 800 μl in 40 ml of assay medium-   5) Dilution of TNF-alpha variants was done as shown below:

Assay medium Final (μl) with concentration 20 ng/ml of wildConcentration in of TNF-alpha in Stock (ul) type TNF-alpha dilutionplate cells K112D: 59 μl 941 100,000 ng/ml 50,000 ng/ml Y115T: 77 μl 923D143K: 32 μl 968 D143R: 34 μl 966 Y115I: 63 μl 937 D143E: 40 μl 960A145R: 50 μl 950 A145K: 50 μl 950 A145E: 26 μl 974 E146K: 40 μl 960E146R: 56 μl 944 500 μl of 500 50,000 ng/ml 25,000 ng/ml 100,000 ng/ml500 μl of 500 25,000 ng/ml 12,500 ng/ml 50,000 ng/ml 400 μl of 60010,000 ng/ml 5000 ng/ml 25,000 ng/ml 500 μl of 500 5,000 ng/ml 2,500ng/ml 10,000 ng/ml 200 μl of 800 1000 ng/ml 500 ng/mL 5000 ng/ml 500 μlof 500 500 ng/ml 50 ng/mL 1000 ng/ml 500 μl of 500 250 ng/ml 125 ng/mLthe 500 ng/ml 400 μl of 600 100 ng/ml 50 ng/mL 250 ng/ml 100 μl of 90010 ng/ml 5 ng/mL 100 ng/ml 100 μl of 900 1 ng/ml 0.5 ng/mL 10 ng/ml 0 00 0

-   5) Dilutions for Inhibition Assay:    -   Stocks to dilute TNF Receptor (TNF R) in 1×assay medium:    -   Stock is 100 μg/ml    -   For 20 μg/ml: 1:5 dilution: 60 μl of 100 μg/ml of Stock+240 μl        of 1×assay medium with wild type TNF-alpha

Dilute TNF R assay medium containing 20 ng/ml of wild type TNF-alpha(final on the cell 10 ng/ml) as shown below:

Assay medium (μl) Final with Concentration Concentration Stock (μl)TNF-alpha in dilution plate in cells 300 μl of 20 μg 300 10,000 ng/ml5000 ng/ml 200 μl of 10,000 ng 300 4000 ng/ml 2000 ng/ml 250 μl of 4000ng 250 2000 ng/ml 1000 ng/ml 250 μl of 2000 ng 250 1000 ng/ml 500 ng/ml 50 μl of 10,000 μg/ml 950 500 ng/ml 250 ng/ml 200 μl of 500 ng/ml 300200 ng/ml 100 ng/ml 100 μl of 500 ng/ml 400 100 ng/ml 50 ng/ml 100 μl of500 ng/ml 900 50 ng/ml 25 ng/ml 200 μl of 50 ng/ml 300 20 ng/ml 10 ng/ml100 μl 50 ng/ml 400 10 ng/ml 5 ng/ml  50 μl 50 ng/ml 450 5 ng/ml 2.5ng/ml  0 250 0 0

All of the above dilutions were done 16 hours prior to adding to thecells. Then 120 μl of each diluted sample was incubated at 4° C., and120 μl of each sample was incubated at 37° C. The next morning, 50 μl ofeach sample was added to the cells. The cells were incubated at 37° C.for 4 hours. After 4 hours of incubation, 100 μl of the caspasesubstrate was added to each well, followed by a 2 hour incubation at 37°C. Read fluorescence.

The results are shown in FIGS. 9 and 10.

Example 4 TNF-alpha Antagonist Activity of Combinatorial TNF-alphaVariants

A) Materials and Method:

-   1) Plate cells for the assay: WEHI164-13Var cells plated at 7.5×105    cells/ml (50 μl/well), incubate at 37 C overnight.-   2) Prepare Assay Media: (10×, final conentration on cells will be 10    ng/mL)    -   7 ml full RPMI    -   5 uL of 310 ug/mL wild type his-TNF [Lot #263-56]    -   140 uL 1 mg/mL ActinomycinD-   3) Dilution of TNF-alpha variants was done as shown below:    -   Mix these samples three days prior to start of experiment

Conc. Final Before Conc. Conc. on Stock (uL) RPMI 10× After 10× cells 1E146K/N34V/V91E (lot 388-3) 1800 ug/mL: 38.6 961.4   69,520 63,200 ng/mL31,600 ng/mL Y115Q/I97T (380-32) 2000 ug/mL: 34.7 965.3   Y115Q/I97R(380-32) 1400 ug/mL: 49.8 950.2   Y115Q/Y87R (380-32) 1100 ug/mL: 63.3936.7   Y115Q/L57Y (380-32) 1100 ug/mL 63.3 936.7   Y115Q/L57F (380-32)1200 ug/mL 57.8 942.2   A145R/L57F (388-3) 2000 ug/mL 34.7 965.3  A145R/Y87H (378-96) 880 ug/mL 78.7 921.3   Enbrel 25000 ug/mL 997.3  Buffer (PBS pH 8) 100 uL 900 TNF R (500 ug/mL) 70 uL 430 2 316 (158 forTNF R) ul of 63,200 ng/mL 684 22,000 20,000 ng/mL 10,000 ng/mL (342) 3316 (158 for TNF R) ul of 20000 ng/mL 684 6,952 6,320 ng/mL 3,160 ng/mL(342) 4 316 (158 for TNF R) ul of 6,320 ng/mL 684 2200 2,000 ng/mL 1000ng/mL (342) 5 316 (158 for TNF R) ul of 2000 ng/mL 684 695.2 362 ng/mL316 ng/mL (342) 6 316 (158 for TNF R) ul of 362 ng/mL 684 220 200 ng/mL100 ng/mL (342) 7 316 (158 for TNF R) ul of 200 ng/mL 684 69.52 63.2ng/mL 31.6 ng/mL (342) 8 316 (158 for TNF R) ul of 63.2 ng/mL 684 22 20ng/mL 10 ng/mL (342) 9 316 (158 for TNF R) ul of 20 ng/mL 684 6.95 6.32ng/mL 3.16 ng/mL (342) 10 316 (158 for TNF R) ul of 6.32 ng/mL 684 2.2 2ng/mL 1 ng/mL (342) 11 316 (158 for TNF R) ul of 2 ng/mL 684 0.69520.632 ng/mL 0.316 ng/mL (342) 12  0 684 0 0 ng/mL 0 (342)

After all dilutions were done add 68.4 (34.2 for TNF R) uL of 10×assaymedia containing WT his TNFa to each dilution well. Then the 96 well wasplaced in the incubator for 3 days. 50 ul of each sample were added toWEHI164-13Var cells for 4 hours. Upon completion of the incubation, add100 ul of caspase substrate. Incubate for 1.5 hours. A R110 curve wasalso prepared by diluting the R110 standard 1:100 in RPMI followed by an8-point half dilution. Then 100 ul of each dilution were added to aplate without cells, these dilutions are done right before adding thesubstrate to the cells. 100 ul of substrate was also added to R110 curvedilutions. Upon the completion of 1.5-hour incubation at 37 C, allsamples were read using the Wallac fluoremeter at 484/535 nmwavelengths.

Results are shown in FIG. 21.

Example 5 Fixed Equilibrium Screening of Many TNF-alpha Variants

Prepare 1:10 fixed equilibrium ratios of TNF-alpha variants:

Mix together 0.01 mg/mL wild type his-TNF [lot #263-56] with 0.1 mg/mLvariant TNF-alpha in 50 uL reactions in phosphate-buffered saline (PBS).

0.33 Conc. Volume mg/mL wt Protein Name Lot# (mg/mL) Prot. (uL) TNF (uL)PBS Y115Q/L57W 380-32 1.3 3.85 1.5 44.65 Y115M/D143N 380-32 0.36 13.81.5 34.7 Y115Q/Y87H 380-32 1.1 4.55 1.5 44 Y115Q/A145R 380-32 0.53 9.41.5 39.1 Y115Q/A145F 380-32 2.0 2.5 1.5 46 Y115Q/L57Y 380-32 1.1 4.551.5 44 Y115M/A145R 380-32 0.74 6.8 1.5 41.7 Y115M/E146K 380-32 0.27 18.51.5 30 Y115M/D143Q 380-32 0.37 13.5 1.5 35 Y115Q/L57F 380-32 1.2 4.171.5 44.3 A145R/I97R 380-32 0.56 9 1.5 39.5 A145R/Y87H 380-32 1.6 3.131.5 45.4 A145R/L75Q 380-32 0.86 5.8 1.5 42.7 A145R/L75K 380-32 0.99 4.91.5 43.6 Y115M/A145R 380-32 0.23 21.7 1.5 27 A145R/S86Q 380-32 1.2 4.21.5 44.3 E146K/V91E/N34E 380-32 1.2 2.8 1.5 45.7 A145R/S86R 378-95 0.2718.5 1.5 30 A145R/I97T 378-97 0.47 10.6 1.5 37.9 A145R/L75E 378-94 1.732.9 1.5 45.6 Y115Q/S86R 380-32 0.94 4.9 1.5 43.6 Y115Q/Y87R 380-32 1.14.6 1.5 43.9 Y115Q/L75K 380-32 0.75 6.7 1.5 41.8 Y115Q/S86Q 380-32 1.04.9 1.5 43.6 Y115Q/E146K 380-32 0.38 13.1 1.5 35.4 Y115Q/L75Q 380-320.58 8.6 1.5 39.9 Y115Q/I97T 380-32 2.0 2.5 1.5 46 Y115Q/D143N 380-320.3 16.7 1.5 31.8 Y115Q/L75E 380-32 0.62 8.1 1.5 40.4 Y115Q/I97R 380-321.4 3.6 1.5 44.9 A145R/L57F 388-3  2 2.5 1.5 46

Prepare this mixture and incubate at 37 C for three-four days

Plate cells for the assay: Human U937 cells plated at 1×106 cells/ml (50μl/well), incubate at 37 C overnight.

-   2) Caspase Assay:

Warm full RPMI medium and supplement with 2 ug/mL Actinomycin D. Mixeach entire 50 uL reaction with 450 uL Actinomycin D supplemented RPMImedium. This mixture is diluted 1:1 eleven times to generate a dosecurve for the fixed equilibrium. 50 uL of the dilution mixture isapplied to the cells in quadruplicate.

Cells are incubated in the TNF-alpha/TNF-alpha variant fixed equilibriumfor 1.5 hours. Upon completion of the incubation, add 100 ul of caspasesubstrate. Incubate for 1.5 hours. A R110 curve was also prepared bydiluting the R110 standard 1:100 in RPMI followed by an 8-point halfdilution. Then 100 ul of each dilution were added to a plate withoutcells, these dilutions are done right before adding the substrate to thecells. 100 ul of substrate was also added to R110 curve dilutions. Uponthe completion of 1.5-hour incubation at 37 C, all samples were readusing the Wallac fluoremeter at 484/535 nm wavelengths.

Results are shown in FIGS. 22A-C.

Example 6 Binding Assay

Biotinylation of TNFa was performed by adding 20 molar excessSulfo-NHS-LC-biotin to the protein sample and incubating the sample onice for 2 hours. Excess biotin was removed from the sample by dialysis.Coupling ratios ranged between 1 to 4. The protein concentration ofbiotinylated TNFa was determined by BCA protein assay (Pierce). Wells ofa microtiter plate were coated with anti-FLAG antibody at aconcentration of 2.5 mg/ml and blocked with 3% BSA overnight at 4° C.The FLAG-tagged protein TNFR1 receptor was added at a concentration of10 ng/ml in PBS+1% BSA to wells of the anti-FLAG-coated microtiterplate, and the plate was incubated for 2 hours at room temperature.Biotinylated TNFa proteins ranging in concentrations from 0-1 mg/mL wereadded in quadruplicate to anti-FLAG-TNFR1-coated wells to representtotal binding. Non-specific binding was measured by adding biotinylatedTNFα proteins ranging in concentrations from 0-1 μg/ml in quadruplicateto wells coated only with anti-FLAG antibody. Binding was allowed tooccur overnight at +4° C. to ensure equilibrium. Alkaline phosphataseconjugated neutravidin (Pierce) was added to the wells at 1:10,000dilution in PBS+1% BSA and incubated for 30 min at room temperature.Luminescence was detected upon the addition of the CSPD star substrate(Applied Biosystems, Foster City, Calif.) and was measured (WallacVICTOR, Perkin Elmer Life Sciences, Boston, Mass.). The specific bindingof TNFa was calculated by subtracting non-specific binding from totalbinding. Data was fit to the binding equation y=(BLmax*x)/(Kd+x).

The results of the binding assays are shown in FIGS. 19A-D. All variantsshow a decrease in receptor binding.

Example 7 7A. TNF-alpha Variants Exchange with Wild Type TNF-alpha toReduce Activation of NFk B

TNF-alpha variants tested were A145R, double variant A145R/Y87H, andtriple variant E146K/V91E/N34E. His-tagged TNF-alpha was pre-incubatedwith 10-fold excess (1:10) of different variants for 3-days at 37degrees C. Wild type TNF-alpha alone and pre-exchanged heterotrimers ofTNF-alpha variants were then tested for their ability to activate anNFkB-driven luciferase reporter (pNFkB-luc, Clontech) in 293T cells.293T cells were seeded at 1.2×104 cells/well in 96-well plates. Cellswere then transfected with pNFkB-luc (NF-kB dependent luciferasereporter) or pTal (Control: basal promoter driving the luciferase gene,but without NFkB binding elements) using Fugene transfection reagentaccording to the manufacturer's protocol (Roche). 12 hrs aftertransfection, cells were treated with a final concentration of 10 ng/mlwild type TNF-alpha or a pre-exchanged mixtures of 10 ng/ml:TNF/100ng/ml variant. 12 hrs after treatment, the cells in 96-well plates wereprocessed for the luciferase assay using the Steady-Glo Luciferase AssaySystem (Promega) according to the manufacturer's protocol. Luminescencefrom each well was measured using the Packard TopCount NXT (PackardBioscience) luminescence counter. Treated samples were tested inquadruplicates, and mean values of luminescence were plotted as barvalues including the standard deviation for each treatment. The resultsare shown in FIG. 20A. The graph shows that the TNF-alpha variants ofthe present invention were effective in decreasing wild-type TNF-alphainduced NFkB activation. The TNF-alpha variant A145R/Y87H was mosteffective in decreasing TNF-alpha induced NFkB activation.

7B. Immuno-localization of NFkB in HeLa Cells

HeLa cells were seeded onto 12 mm sterile coverslips (Fisherbrand) at adensity of 1.5×105 cells/well in 6-well plates and cultured at 37degrees C. at 5% CO2 atmosphere. The following day, the cells weretreated with various concentrations of his-tagged wild type TNF-alpha,A145R/Y87H variant alone, or the combination of the his-tagged TNF-alphaand 10-fold excess of the A145/Y87H variant (pre-exchanged for threedays at 37 C) at 37° C., 5% CO2. After 30 minutes of incubation, thecells attached to coverslips in 6-well plates were briefly washed withPBS and fixed in 4% formaldehyde/PBS for 10 minutes. Cells were thenwashed an additional five times with PBS or maintained in the last PBSwash overnight before processing cells for immunocytochemistry. Fixedcells on coverslips were then treated with 0.1% Triton X-100/PBS. Thebuffer was aspirated and cells on coverslips were blocked in ahumidified chamber for 15 minutes with 50 ul of 0.1% BSA/0.1% TX-100/PBSper coverslip at 37° C. The blocking reagent was then removed andreplaced with primary antibody against p65 subunit of NF-kB (pAb C-20,Santa Cruz Bioscience). After one hour of incubation at 37 degrees C.,the antibody was removed and coverslips were washed 5 times with PBS. 50ul of FITC-conjugated secondary antibody (Jackson Immuno laboratories)diluted in blocking buffer (1:100) was added to each coverslip (JacksonImmunolaboratories) and coverslips were incubated in a light-safehumidified chamber for an additional hour before removing the secondaryantibody with 5 washes of PBS. Coverslips were briefly rinsed withd-water, air-dried in a light-safe chamber and mounted onto slides usingAnti-fade (Molecular Probes). Digital images of antibody-reacted cellswere captured using a FITC filter and 40× objective on a Nikon EclipseTS100 microscope coupled to a Cool SNAP-Pro CCD camera (MediaCybernetics) and operated using Image Pro Plus software (MediaCybernetics).

FIG. 20B shows photographs of the immuno-localization of NFkB in HeLacells showing that the exchange of wild type TNF-alpha with theA145/Y87H TNF-alpha variant inhibits TNF-alpha-induced nucleartranslocation of NFkB in HeLa cells. The TNF-alpha variant A145R/Y87Halone does not induce NFkB nuclear translocation, unlike the wild-typeTNF-alpha. Moreover, the wild type TNF-alpha exchanged (3-days, 37degrees C.) to form heterotrimers with excess variant (10 fold excess ofTNF-alpha variant A145R/Y87H) loses its ability to induce NFkB nucleartranslocation. This data is consistent with the effects of this variantin the luciferase reporter assay.

7C. TNF-Alpha Variant A145R/Y87H Reduced TNF-alpha Induced Activation ofthe NFkB-driven Luciferase Reporter

His-tagged wild type TNF-alpha, TNF-alpha variant A145/Y87H and theexchanged wild type TNF-alpha:A145R/Y87H heterotrimer (1-day exchangewith 10-fold excess TNF-alpha variant A145R/Y87H at 37 degrees C.) weretested in the NFkB luciferase reporter assay as in Example 7A above.

The experiment was carried out as in Example 7A, with the exception thata wider range of final TNF-alpha concentrations and increasing doseswere used (0.78, 1.56, 3.13, 6.25, 12.5, 25 ng/ml) with 10-fold excessof TNF-alpha variant (A145R/Y87H) at each TNF-alpha concentration.

The wild type TNF-alpha:A145R/Y87H heterotrimer has a significantlyreduced activation level, indicating the TNF-alpha A145R/Y87H variant'sinhibitory effect on wild type TNF-alpha. Unlike wild type TNF-alpha,the TNF-alpha variant A145/Y87H alone has no significant agonizingeffect on NFkB activation as shown by the lower dotted line in FIG. 20C.Wild type TNF-alpha induced activation is dependent on the NFkBactivation as the reporter and without NFkB binding elements isunresponsive to the TNF-alpha as shown in the solid gray line in FIG.20C.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

1. A variant Tumor necrosis factor α (TNF-alpha) protein comprising anamino acid sequence that has at least one amino acid substitution in theLarge Domain and at least one amino acid substitution in a domainselected from the group consisting of the DE Loop and the Small Domainas compared to the wild type TNF-alpha sequence of amino acids 1-157 ofSEQ ID NO:2, wherein the Large Domain substitution is at a positionselected from the group consisting of 21, 30, 31, 32, 33, 35, 65, 66,67, 111, 112, 115, 140, 143, 144, 145 and 146, wherein the Small Domainsubstitution at a position selected from the group consisting of 75 and97, wherein the DE Loop substitution at a position selected from thegroup consisting of 84, 86, 87 and 91, and wherein said variantTNF-alpha protein is capable of interacting with the wild type TNF-alphato form mixed trimers having at least a 50% decrease in receptoractivation as compared to a homotrimer of wild-type TNF-alpha proteinsas determined by a caspase assay.
 2. A variant TNF-alpha proteinaccording to claim 1, wherein said variant TNF-alpha protein has from 2to 5 amino acid substitutions as compared to wild type TNF-alphasequence.
 3. A variant TNF-alpha protein according to claim 1, whereinsaid Large Domain substitution is at position
 145. 4. A variantTNF-alpha protein according to claim 1, further comprising at least oneadditional substitution in the Trimer Interface of said TNF-alphaprotein at a position selected from the group consisting of
 34. 91 and57.
 5. A variant TNF-alpha protein according to claim 1, wherein saidvariant TNF-alpha protein is capable of interacting with the wild typeTNF-alpha to form mixed trimers having at least a 76% decrease inreceptor activation as compared to a homotrimer of wild-type TNF-alphaproteins as determined by a caspases assay.
 6. A variant TNF-alphaprotein according to claim 5, wherein said variant TNF-alpha protein iscapable of interacting with the wild type TNF-alpha to form mixedtrimers having at least a 90% decrease in receptor activation ascompared to a homotrimer of wild-type TNF-alpha proteins as determinedby a caspase assay.
 7. A variant TN F-alpha protein according to claim5, wherein said variant TNF-alpha protein is capable of interacting withthe wild type TNF-alpha to form mixed trimers that are incapable ofactivating receptor signaling as determined by a caspase assay.
 8. Avariant TNF-alpha protein according to claim 1, wherein said LargeDomain substitution is selected from the group of substitutionsconsisting of Q21C, Q2IR, Q21R, N30D, R31C, R31I, R31D, R31E, R32D,R32E, R32S, A33E, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V,K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S. Q67W, Q67Y, A111R, A111E,K112D, K112E, Y115D, Y115E, Y115F, Y115H, Y1151, Y115K, Y115L, Y115M,Y115N, Y115Q, Y115R, Y115S, Y115T, Y115W, D140K, D140R, D143E, D143K,D143L, D143R, D143N, D143Q, D143R, D143S, F144N, A145D, A145E, A145F,A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, A145Y, E146K,E146L, E146M, E146N, E146R, E146S and S147R, and said DE Loopsubstitution is selected from the group consisting of A84V, S86Q, S86R,Y87H, Y87R and V91E.
 9. A variant protein according to claim 8, whereinsaid Large Domain substitution is selected from the group consisting ofY115I, Y115Q, Y115T, D143K, D143R, D143E, A145E, A145R, E146K and E146R.10. A variant protein according to claim 8, wherein said substitutionscomprise (a) either Y151I or Y115T and (b) either A145E or A145R.
 11. Avariant protein according to claim 8, wherein said substitutionscomprise A145R and further comprise at least one of L57F, L75E, L75Q,Y87H, or I97T.
 12. A variant protein according to claim 11, wherein saidsubstitutions comprise A144R and Y87H.
 13. A variant protein accordingto claim 8, wherein said substitutions comprise L57F and A145E.
 14. Avariant protein according to claim 1, wherein said Large Domainsubstitution Is selected from the group consisting of Y115I, Y115T,D143K, D143R, D143E, A145R, E146K and E146R.
 15. A variant proteinaccording to claim 1, wherein from about 1 to about 55 amino acids fromthe N-terminus, the C-terminus or both the N and C termini of saidprotein are deleted.
 16. A variant protein according to claim 15 whereinresidues on the N-terminus are deleted beyond residue
 10. 17. A variantprotein according to claim 16 wherein the first 47 N-terminal aminoacids are deleted.
 18. A variant protein of claim 17 wherein theC-terminal leucine is deleted.
 19. A pharmaceutical compositioncomprising a variant TNF-alpha protein according to claim 1 and apharmaceutical carrier.
 20. A variant amino acid according to claim 1,wherein said Large Domain substitution is at a position selected fromthe group consisting of positions 21, 30, 31, 32, 33, 35, 65, 66, 67,111, 112, 115, 140, 143, 144, 145 and 146, said Small Domainsubstitution is at a position selected from the group consisting ofpositions 75 and 97, and said DE Loop substitution is at a positionselected from the group consisting of positions 84, 88, 87 and
 91. 21. Avariant Tumor necrosis factor α (TNF-alpha) comprising an amino acidsequence that has at least one amino acid substitution in the LargeDomain and at least one amino acid substitution in a domain selectedfrom the group consisting of the DE Loop and the Small Domain ascompared to the wild type TNF-alpha sequence of amino acids 1-157 of SEQID NO:2, wherein the Large Domain substitution is at a position selectedfrom the group consisting of 21, 30, 31, 32, 33, 35, 65, 66, 67, 111,112, 115, 140, 143, 144, 145 and 146, wherein the DE Loop substitutionat a position selected from the group consisting of 84, 86, 87 and 91,wherein the Small Domain substitution at a position selected from thegroup consisting of 75 and and 97, and wherein said variant TNF-alphaprotein forms mixed trimers are capable of interacting with a receptorinterface at the receptor binding site to reduce receptor activation byat least 50% as compared to a homotrimer of wild-type TNF-alpha proteinsas determined by a caspase assay.
 22. A variant TNF-alpha proteinaccording to claim 21, wherein said variant TNF-alpha protein blocks thereceptor binding site.
 23. A variant protein according to claims 1 or 21wherein said protein is PEGylated.
 24. A variant protein according toclaim 23, wherein said protein is PEGylated at positions selected fromthe group consisting of 10, 21 23, 24, 25, 27, 31, 42, 44, 45, 46, 86,87, 88, 90, 107, 108, 128, 110, 140 and
 145. 25. A variant proteinaccording to claim 24, wherein said protein is PEGylated at positionsselected from the group consisting of 21, 23, 31 and
 45. 26. A variantprotein according to claim 25, wherein said protein is PEGylated at theN-terminus.
 27. A variant protein according to claim 23 wherein saidPEGylated protein disrupts receptor binding.
 28. A variant protein ofclaim 1 or 21 wherein said protein is circularly permuted or cyclized.29. A variant protein according to claim 1 or 21 wherein two or moremodified domains are covalently linked via disulfide bonds.
 30. Avariant protein according to claim 1 or 21, wherein two or more modifieddomains are covalently linked via chemical cross linking.
 31. A variantprotein according to claim 1 or 21, wherein two or more TNF-alphavariant proteins are covalently linked by a linker peptide.
 32. Avariant protein according to claim 31, wherein said linker peptide is asequence of at least one and not more than about 30 amino acid residues.33. A variant protein according to claim 32, wherein said linker peptideis a sequence of at least 5 and not more than about 20 amino acidresidues.
 34. A variant protein according to claim 33, wherein saidlinker peptide is a sequence of at least 10 and not more than about 15amino acid residues.
 35. A variant protein according to claim 31,wherein the linker peptide comprises one or more of the following aminoacid residues: Gly, Ser, Ala, or Thr.
 36. A variant protein according toclaim 1 or 21, further comprising an M at position 1.